U.S. patent application number 12/062412 was filed with the patent office on 2010-11-25 for highly sensitive system and methods for analysis of troponin.
Invention is credited to Philippe J. Goix, Douglas Held, Richard A. Livingston, Robert Puskas, John Todd, Alan H.B. Wu.
Application Number | 20100297672 12/062412 |
Document ID | / |
Family ID | 46330235 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297672 |
Kind Code |
A9 |
Goix; Philippe J. ; et
al. |
November 25, 2010 |
HIGHLY SENSITIVE SYSTEM AND METHODS FOR ANALYSIS OF TROPONIN
Abstract
The invention provides methods, compositions, kits, and systems
for the sensitive detection of cardiac troponin. Such methods,
compositions, kits, and systems are useful in diagnosis, prognosis,
and determination of methods of treatment in conditions that
involve release of cardiac troponin.
Inventors: |
Goix; Philippe J.; (Oakland,
CA) ; Puskas; Robert; (Manchester, MO) ; Todd;
John; (Lafayette, CA) ; Livingston; Richard A.;
(Webster Groves, MO) ; Held; Douglas; (Ballwin,
MO) ; Wu; Alan H.B.; (Palo Alto, CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20080261242 A1 |
October 23, 2008 |
|
|
Family ID: |
46330235 |
Appl. No.: |
12/062412 |
Filed: |
April 3, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11784213 |
Apr 4, 2007 |
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12062412 |
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60914995 |
Apr 30, 2007 |
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60925402 |
Apr 19, 2007 |
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60789304 |
Apr 4, 2006 |
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60793664 |
Apr 19, 2006 |
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60808622 |
May 26, 2006 |
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60861498 |
Nov 28, 2006 |
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60872986 |
Dec 4, 2006 |
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Current U.S.
Class: |
435/7.21 ;
436/536 |
Current CPC
Class: |
G01N 33/6887 20130101;
G01N 21/6486 20130101; G01N 21/6428 20130101; G01N 33/582 20130101;
G01N 33/6893 20130101; G01N 2800/325 20130101; G01N 2333/47
20130101; G01N 2800/32 20130101; G01N 33/68 20130101; G01N 2800/50
20130101; G01N 2800/324 20130101 |
Class at
Publication: |
435/7.21 ;
436/536 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/536 20060101 G01N033/536 |
Claims
1. A composition comprising a label for cardiac troponin comprising
a detection binding partner for cardiac troponin I, wherein the
detection binding partner is capable of cross-reacting with cardiac
troponin I from at least two species, and a fluorescent moiety,
wherein said moiety is capable of emitting at least about 200
photons when simulated by a laser emitting light at the excitation
wavelength of the moiety, wherein the laser is focused on a spot
not less than about 5 microns in diameter that contains the moiety,
and wherein the total energy directed at the spot by the laser is
no more than about 3 microJoules.
2. The composition of claim 1 wherein the detection binding partner
is capable of reacting with cardiac troponin I from at least two
species selected from the group consisting of human, monkey, dog,
and rat.
3. The composition of claim 2 wherein the detection binding partner
is capable of reacting with cardiac troponin I from human, monkey,
dog, and rat.
4. The composition of claim 1 wherein the detection binding partner
is an antibody.
5. The composition of claim 1 further comprising a capture binding
partner for cardiac troponin I, wherein the capture binding partner
is capable of cross-reacting with cardiac troponin I from at least
two species.
6. The composition of claim 5 wherein the capture binding partner
is capable of reacting with cardiac troponin I from at least two
species selected from the group consisting of human, monkey, dog,
and rat.
7. The composition of claim 5 wherein the capture binding partner
is capable of reacting with cardiac troponin I from human, monkey,
dog, and rat.
8. The composition of claim 5 wherein the capture binding partner
is an antibody.
9. A method of determining a course of action for an individual
comprising: i) obtaining a sample from said individual, ii)
detecting the level of cardiac troponin in the sample and iii) if
the level of cardiac troponin is more than about 10-fold greater
than the 99.sup.th percentile normal value for cardiac troponin,
taking a first action with respect to the individual and if the
level of cardiac troponin is not more than about 10-fold of the
99.sup.th percentile value for cardiac troponin, taking a second
action.
10. The method of claim 9 wherein the individual is a patient being
evaluated for a possible cardiac event or cardiotoxicity.
11. The method of claim 9 wherein the first action is admission of
the individual to a hospital.
12. The method of claim 9 wherein the second action is holding said
individual for a period of time and taking a series of samples from
said patient over said period of time.
13. The method of claim 12 wherein the interval between samples is
at less than about 4 hours.
14. The method of claim 12 wherein the rate of change in the level
of troponin between individual samples in the series of samples is
detected.
15. The method of claim 14 wherein the change in the level of
troponin is a decrease.
16. The method of claim 14 wherein the change in the level of
troponin is an increase.
17. The method of claim 14 wherein a decision is made regarding a
course of action for said individual based on said rate of
change.
18. The method of claim 14 wherein if the rate of change in
troponin values exceeds a predetermined upper reference rate of
change value, the decision is to admit the individual.
19. The method of claim 14 wherein a decision to take a first
action is made if one or more spikes in the level of troponin is
seen from sample to sample.
20. The method of claim 14 wherein the change in the level of
troponin indicates an acute cardiovascular disease.
21. The method of claim 14 wherein the change in the level of
troponin indicates a chronic cardiovascular disease.
22. The method of claim 14 wherein the change in the level of
troponin indicates cardiotoxicity.
23. The method of claims 9 wherein the cardiac troponin is cardiac
troponin I.
Description
CROSS REFERENCE
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Application 60/914,995, filed Apr. 30, 2007,
and U.S. Provisional Application 60/925,402, filed Apr. 19, 2007.
This application also claims priority under 35 U.S.C. .sctn. 119 to
U.S. Provisional Application No. 60/789,304, filed Apr. 4, 2006,
U.S. Provisional Application No. 60/861,498, filed Nov. 28, 2006,
and U.S. Provisional Application No. 60/872,986, filed Dec. 4,
2006, all of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Each year in the United States, some six million people
present to emergency departments with chest pain. Although only 15%
to 20% of these patients are ultimately diagnosed with an acute
coronary syndrome (ACS), about half are admitted for evaluation.
Conversely, 2% of patients with ACS are mistakenly discharged. As
patients with ACS have a relatively high risk of major adverse
cardiovascular events in the short term, there is a clear need for
accurate objective tools by which to identify them.
[0003] Currently used markers for cardiac damage suffer
disadvantages that limit their clinical usefulness. Cardiac enzyme
assays have formed the basis for determining whether or not there
is damage to the cardiac muscle. Unfortunately, the standard
creatine kinase-MB (CK-MB) assay is not reliable in excluding
infarction until 10 to 12 hours after the onset of chest pain.
Earlier diagnosis would have very specific advantages with regard
to fibrinolytic therapy and triage. In additional, more sensitive
markers of cardiac toxicity would allow earlier detection of
adverse effects of, e.g., drug therapy.
SUMMARY OF THE INVENTION
[0004] In one aspect the invention provides methods.
[0005] In some embodiments, the invention provides a method for
determining the presence or absence of a single molecule of
troponin or a fragment or complex thereof in a sample, including i)
labeling the molecule, fragment, or complex, if present, with a
label; and ii) detecting the presence or absence of the label,
where the detection of the presence of the label indicates the
presence of the single molecule, fragment, or complex of troponin
in the sample. In some embodiments of the methods of the invention,
the troponin is a cardiac isoform of troponin. In some embodiments
of the methods of the invention, the troponin can be cardiac
troponin I (cTnI) or cardiac troponin C (cTnC). In some embodiments
of the methods of the invention, the troponin is cTnI. In some
embodiments of the methods of the invention, a single molecule of
troponin can be detected at a limit of detection of less than about
100 pg/ml. In some embodiments of the methods of the invention, a
single molecule or troponin can be detected at a level of detection
of less than about 20 pg/ml. In some embodiments of the methods of
the invention, the label includes a fluorescent moiety. In some
embodiments, the fluorescent moiety is capable of emitting at least
about 200 photons when simulated by a laser emitting light at the
excitation wavelength of the moiety, where the laser is focused on
a spot not less than about 5 microns in diameter that contains the
moiety, and where the total energy directed at the spot by the
laser is no more than about 3 microJoules. In some embodiments of
the methods of the invention, the fluorescent moiety includes a
molecule that contains at least one substituted indolium ring
system in which the substituent on the 3-carbon of the indolium
ring contains a chemically reactive group or a conjugated substance
group. In some embodiments of the methods of the invention, the
fluorescent moiety includes a dye. Examples of dyes include, but
are not limited to, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647,
AlexaFluor 680 and AlexaFluor 700. In some embodiments of the
methods of the invention, the fluorescent moiety includes
AlexaFluor 647. In some embodiments, the fluorescent moiety
includes a molecule that contains at least one substituted indolium
ring system in which the substituent on the 3-carbon of the
indolium ring contains a chemically reactive group or a conjugated
substance group. In some embodiments of the methods of the
invention, the label further includes a binding partner for the
troponin molecule, fragment, or complex. In some embodiments of the
methods of the invention, the binding partner includes an antibody
specific to the troponin molecule, fragment, or complex. In some
embodiments of the methods of the invention, the antibody is
specific to a specific region of the troponin molecule. In some
embodiments of the methods of the invention, the antibody is
specific to a region comprising amino acids 27-41 of cardiac
troponin I. In some embodiments of the methods of the invention,
the antibody can be a polyclonal antibody. In some embodiments of
the methods of the invention, the antibody is a monoclonal
antibody. In some embodiments of the methods of the invention, the
methods further include capturing troponin or troponin complex on a
solid support. In some embodiments of the methods of the invention,
the solid support can be a microtiter plate or paramagnetic beads.
In some embodiments of the methods of the invention, the solid
support includes a capture partner specific for the troponin or
troponin complex that is attached to the solid support. In some
embodiments of the methods of the invention, the attachment of the
capture partner to the solid support is noncovalent. In some
embodiments of the methods of the invention, the attachment of the
capture partner to the solid support is covalent. In some
embodiments of the methods of the invention, the covalent
attachment of the capture partner is such that the capture partner
is attached to the solid support in a specific orientation. In some
embodiments of the methods of the invention, the specific
orientation serves to maximize specific binding of the troponin or
troponin complex to the capture partner. In some embodiments of the
methods of the invention, the capture partner comprises an
antibody. In some embodiments of the methods of the invention, the
antibody is a monoclonal antibody. In some embodiments of the
methods of the invention, antibody is specific to amino acids 87-91
of cardiac troponin I. In some embodiments of the methods of the
invention, the antibody is specific to amino acids 41-49 of cardiac
troponin I. In some embodiments of the methods of the invention,
the sample is a blood, serum, or plasma sample. In some embodiments
of the methods of the invention, the sample is a serum sample. In
some embodiments of the methods of the invention, the label include
a fluorescent moiety, and step ii) includes passing the label
through a single molecule detector. In some embodiments of the
methods of the invention, the single molecule detector include: a)
an electromagnetic radiation source for stimulating the fluorescent
moiety; b) a capillary flow cell for passing the fluorescent
moiety; c) a source of motive force for moving the fluorescent
moiety in the capillary flow cell; d) an interrogation space
defined within the capillary flow cell for receiving
electromagnetic radiation emitted from the electromagnetic source;
e) an electromagnetic radiation detector operably connected to the
interrogation space for measuring an electromagnetic characteristic
of the stimulated fluorescent moiety; and f) a microscope objective
lens situated between the interrogation space and the detector,
where the lens is a high numerical aperture lens.
[0006] In some embodiments, the invention provides a method for
determining a diagnosis, prognosis, or method of treatment in an
individual that includes: i) determining a concentration of cardiac
troponin in a sample or determining the concentrations of cardiac
troponin in a series of samples from the individual, where the
concentration is determined by a cardiac troponin assay with a
limit of detection for the cardiac troponin in the sample of less
than about 50 pg/ml; and ii) determining a diagnosis, prognosis, or
method of treatment in the individual, based on the concentration
in the sample, or on the concentrations in the series of samples.
In some embodiments of the methods of the invention, step ii)
includes an analysis such as comparing the concentration or series
of concentrations to a normal value for the concentration,
comparing the concentration or series of concentrations to a
predetermined threshold level, comparing the concentration or
series of concentrations to a baseline value, and determining a
rate of change of concentration for the series of concentrations.
In some embodiments of the methods of the invention, step ii)
includes comparing the concentration of troponin in the sample with
a predetermined threshold concentration, and determining a
diagnosis, prognosis, or method of treatment if the sample
concentration is greater than the threshold level. In some
embodiments of the methods of the invention, the threshold
concentration is determined by determining a the 99th percentile
concentration of troponin in a group of normal individuals, and
setting the threshold concentration at the 99th percentile
concentration. In some embodiments of the methods of the invention,
at least one sample is taken during or after a cardiac stress test.
In some embodiments of the methods of the invention, the cardiac
troponin is selected from the group consisting of cardiac troponin
I and cardiac troponin T. In some embodiments of the methods of the
invention, the cardiac troponin is cardiac troponin I. In some
embodiments of the methods of the invention, the concentration of
cardiac troponin is a concentration of total cardiac troponin. In
some embodiments of the methods of the invention, the concentration
of cardiac troponin is a concentration of a cardiac troponin
complex, cardiac troponin fragment, phosphorylated cardiac
troponin, oxidized cardiac troponin, or a combination thereof. In
some embodiments of the methods of the invention, the concentration
of cardiac troponin is compared to total cardiac troponin. In some
embodiments of the methods of the invention, the diagnosis,
prognosis, or method of treatment is a diagnosis, prognosis, or
method of treatment of myocardial infarct. In some embodiments of
the methods of the invention, the diagnosis, prognosis, or method
of treatment comprises risk stratification for level of risk of
myocardial infarct. In some embodiments of the methods of the
invention, the concentration or series of concentrations is
determined at or near the time the individual presents to a health
professional with one or more symptoms indicative of myocardial
ischemia or infarct or the possibility thereof. In some
embodiments, the one or more symptoms can be chest pain, chest
pressure, arm pain, abnormal EKG, abnormal enzyme levels, or
shortness of breath. In some embodiments, the concentration is
determined by a method that includes detecting single molecules of
troponin, or complexes or fragments thereof. In some embodiments,
the methods of the invention involve labeling troponin or a
troponin complex with a label that comprises a fluorescent moiety.
In some embodiments of the methods of the invention, the
fluorescent moiety is capable of emitting at least about 200
photons when simulated by a laser emitting light at the excitation
wavelength of the moiety, where the laser is focused on a spot 5
microns in diameter that contains the moiety, and where the total
energy directed at the spot by the laser is no more than about 3
microJoules. In some embodiments of the methods of the invention,
the fluorescent moiety includes a molecule that contains at least
one substituted indolium ring system in which the substituent on
the 3-carbon of the indolium ring contains a chemically reactive
group or a conjugated substance group. In some embodiments of the
methods of the invention, the fluorescent moiety includes a dye
selected from the group consisting of AlexaFluor 488, AlexaFluor
532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. In some
embodiments of the methods of the invention, the fluorescent moiety
comprises AlexaFluor 647. In some embodiments of the methods of the
invention, the label further comprises a binding partner for the
troponin. In some embodiments, the binding partner comprises an
antibody specific to the troponin. In some embodiments, the
antibody is a polyclonal antibody. In some embodiments of the
methods of the invention, the methods further include capturing
troponin or troponin complex on a solid support. In some
embodiments of the methods of the invention, the solid support can
be a microtiter plate or paramagnetic beads. In some embodiments of
the methods of the invention, the solid support includes a capture
partner specific for the troponin or troponin complex that is
attached to the solid support. In some embodiments of the methods
of the invention, the attachment of the capture partner to the
solid support is noncovalent. In some embodiments of the methods of
the invention, the attachment of the capture partner to the solid
support is covalent. In some embodiments of the methods of the
invention, the covalent attachment of the capture partner is such
that the capture partner is attached to the solid support in a
specific orientation. In some embodiments of the methods of the
invention, the specific orientation serves to maximize specific
binding of the troponin or troponin complex to the capture partner.
In some embodiments of the methods of the invention, step i)
further involves assessing another indicator for the individual,
and step ii) involves determining a diagnosis, prognosis, or method
of treatment in the individual, based on the concentration of
troponin and the assessment of the other indicator of the
non-troponin marker in the sample, or on the concentrations in the
series of samples. In some embodiments, the other indicator is a
clinical indicator of myocardial ischemia or infarct. In some
embodiments, the other indicator is the concentration of one or
more non-troponin markers in the sample or the series of samples.
In some embodiments of the methods of the invention, the one or
more markers are markers of cardiac ischemia, or markers of
inflammation and of plaque instability. In some embodiments, the
one or more markers of cardiac ischemia can be creatine kinase (CK)
and its myocardial fraction CK myocardial band (MB), aspartate
aminotransferase, lactate dehydrogenase (LDH),
.alpha.-hydroxybutyrate dehaydrogenase, myoglobin, glutamate
oxaloacetate transaminase, glycogen phosphorylase BB, unbound free
fatty acids, heart fatty acid binding protein (H-FABP),
ischemia-modified albumin, myosin light chain 1, or myosin light
chain 2. In some embodiments of the methods of the invention, the
one or more markers include one or more specific markers of
myocardial injury. In some embodiments of the methods of the
invention, the diagnosis, prognosis, or method of treatment is a
diagnosis, prognosis, or method of treatment of a condition that is
not myocardial infarct. In some embodiments, the condition is
cardiac toxicity. In some embodiments, the cardiac toxicity is
associated with the administration of a drug to the individual. In
some embodiments of the methods of the invention, the condition is
selected from the group consisting of acute rheumatic fever,
amyloidosis, cardiac trauma (including contusion, ablation, pacing,
firing, cardioversion, catheterization and cardiac surgery),
reperfusion injury, congestive heart failure, end-stage renal
failure, glycogen storage disease type II (Pompe's disease), heart
transplantation, haeomoglobinopathy with transfusion
haemosiderosis, hypertension, including gestational hypertension,
hypotension, often with arrhythmias, hypothyroidism, myocarditis,
pericarditis, post-operative non-cardiac surgery, pulmonary
embolism, and sepsis.
[0007] In some embodiments, provided is a method of classifying an
individual having one or more symptoms of cardiac infarct
comprising: i) obtaining a sample from said individual, ii)
detecting the level of troponin in the sample and iii) if the level
of troponin is above 10-fold of the 99th percentile, taking a first
action with respect to the individual and if the level of troponin
is below 10-fold of the 99th percentile taking a second action. In
some embodiments, the first action is taken if patient has about a
3-fold to about a 10-fold increase relative to the 99th percentile
cutoff. In some embodiments, the first action is taken if patient
has about a 4-fold to about a 5-fold increase relative to the 99th
percentile cutoff. In some embodiments, the first action is taken
if patient has about a 6-fold to about a 7-fold increase relative
to the 99th percentile cutoff. In some embodiments, the first
action is taken if patient has about an 8-fold to about a 10-fold
increase relative to the 99th percentile cutoff. In some
embodiments, the first action is taken if patient has about a
1.5-fold to about a 2-fold increase. In some embodiments, the first
action is taken if patient has about a 2-fold to about a 2.5-fold
increase. In some embodiments, the first action is taken if patient
has about a 2.5-fold to about a 3-fold increase. In some
embodiments, the first action is taken if patient has about a
3-fold to about a 3.5-fold increase. In some embodiments, the first
action is taken if patient has about a 3.5-fold to about a 4-fold
increase. In some embodiments, the first action is taken if patient
has about a 4-fold to about a 4.5-fold increase. In some
embodiments, the first action is taken if patient has about a
4.5-fold to about a 5-fold increase. In some embodiments, the first
action is taken if patient has about a 5-fold to about a 5.5-fold
increase. In some embodiments, the first action is taken if patient
has about a 5.5-fold to about a 6-fold increase. In some
embodiments, the first action is taken if patient has about a
6-fold to about a 6.5-fold increase. In some embodiments, the first
action is taken if patient has about a 6.5-fold to about a 7-fold
increase. In some embodiments, the first action is taken if patient
has about a 7-fold to about a 7.5-fold increase. In some
embodiments, the first action is taken if patient has about a
7.5-fold to about an 8-fold increase. In some embodiments, the
first action is taken if patient has about a 8-fold to about a
8.5-fold increase. In some embodiments, the first action is taken
if patient has about an 8.5-fold to about a 9-fold increase. In
some embodiments, the first action is taken if patient has about a
9-fold to about a 9.5-fold increase. In some embodiments, the first
action is taken if patient has about a 9.5-fold to about a 10-fold
increase. In some embodiments the first action is taken if the
increase is more than about 2-fold. In some embodiments the first
action is taken if the increase is more than about 3-fold. In some
embodiments the first action is taken if the increase is more than
about 4-fold. In some embodiments the first action is taken if the
increase is more than about 5-fold. In some embodiments the first
action is taken if the increase is more than about 6-fold. In some
embodiments the first action is taken if the increase is more than
about 7-fold. In some embodiments the first action is taken if the
increase is more than about 8-fold. In some embodiments the first
action is taken if the increase is more than about 9-fold. In some
embodiments the first action is taken if the increase is more than
about 10-fold.
[0008] In some embodiments, the individual is a patient being
evaluated for a possible cardiac event. In such a case, the first
action can be admission of the individual to a hospital, or other
appropriate clinical action for treatment of the cardiac event. The
second action can be to hold the individual for a period of time
for further observation. During the time the individual is held a
series of samples are taken from said patient and the level of
troponin in each sample detected. In some embodiments, the interval
between samples is less than about 4, 3, 2, 1, 0.5 hours apart. In
some embodiments, the sample interval is less than about 4 hours
apart. In some embodiments, the sample interval is about 1 hour. In
some embodiments, the sample interval is about 2 hours. It will be
recognized that circumstances in the clinic dictate sample
intervals and some degree of variation is acceptable and is
encompassed within the invention, as long as results may be
reliably interpreted. In some embodiments, in addition to the level
of troponin detected in each sample, the rate of change in the
level of troponin over two or more samples in the series of samples
is detected. In some embodiments, the change in the level of
troponin is a decrease. In some embodiments, the change in the
level of troponin is an increase. A decision can be made regarding
a course of action for said individual based on said rate of
change, e.g., if the rate of change in troponin values exceeds a
predetermined upper reference rate of change value, the decision is
to admit the individual. It will be recognized that other clinical
factors well-known to those of skill in the art can be used to
modify the decision based on troponin levels, e.g., if other
clinical manifestations of acute myocardial infarct are present,
such as abnormal EKG, abnormal enzyme levels, physical symptoms,
and the like, then the threshold for troponin that leads to an
admission is suitably decreased. The decision to take a first
action, e.g., to admit a patient, can also be made if one or more
spikes in the level of troponin are seen from sample to sample. The
spike can be any increase in the level of troponin detected
compared to one or more samples wherein the increase is compared to
a threshold level. For example, if the threshold level is
determined to be 10 pg/ml and if in one of the sample of the series
of samples the troponin level detected is 15 pg/ml, the patient is
admitted. In some embodiments, the threshold level is determined
from a reference population. In some embodiments, the threshold
level is set at a level for an individual, e.g., the initial level
of troponin detected from the first sample taken from the patient.
In these embodiments, a relative increase indicates a spike, no
matter what the absolute values are. In some embodiments, a spike
is determined when the troponin level increases 2, 3, 4, 5, 6, 7,
8, 9 10, or more than 10-fold above a reference value for the
individual, e.g., a value set at the troponin level in the first
sample taken from the individual. Alternatively, a spike in the
level of troponin can be seen when comparing troponin levels
between samples from the individual. In such a case, no matter what
the value of the level of troponin detected, the patient is
admitted if the value increases by more than a certain amount,
e.g., a patient is admitted when the level of troponin increases by
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 pg/ml as compared to
one or more neighboring samples. In some embodiments, the change in
the level of troponin indicates an acute cardiovascular disease. In
some embodiments, the change in the level of troponin indicates a
chronic cardiovascular disease. In some embodiments, the change in
the level of troponin indicates cardiotoxicity.
[0009] In another aspect the invention includes compositions.
[0010] In some embodiments the invention includes a composition for
the detection of a troponin isoform including a binding partner to
the troponin isoform attached to a fluorescent moiety, where the
fluorescent moiety is capable of emitting at least about 200
photons when simulated by a laser emitting light at the excitation
wavelength of the moiety, where the laser is focused on a spot not
less than about 5 microns in diameter that contains the moiety, and
where the total energy directed at the spot by the laser is no more
than about 3 microJoules. In some embodiments of the compositions
of the invention, the binding partner comprises an antibody to the
troponin isoform. In some embodiments, the antibody is a polyclonal
antibody. In some embodiments, the antibody is a monoclonal
antibody. In some embodiments, the troponin isoform is a cardiac
isoform. In some embodiments, the cardiac isoform is selected from
the group consisting of cTnI and cTnT. In some embodiments, the
cardiac isoform is cTnI. In some embodiments, the antibody is
specific to a specific region of the troponin molecule. In some
embodiments, the antibody is specific to a region comprising amino
acids 27-41 of cardiac troponin I. In some embodiments of the
compositions of the invention, the fluorescent moiety comprises a
molecule that comprises at least one substituted indolium ring
system in which the substituent on the 3-carbon of the indolium
ring contains a chemically reactive group or a conjugated substance
group. In some embodiments, the fluorescent moiety includes a dye
that can be AlexaFluor 488, AlexaFluor 532, AlexaFluor 647,
AlexaFluor 680 or AlexaFluor 700. In some embodiments, the
fluorescent moiety comprises AlexaFluor 647.
[0011] In some embodiments the invention involves a composition
comprising a set of standards for the determination of a
concentration of a cardiac troponin, where at least one of the
standards is at a concentration of cardiac troponin less than about
10 pg/ml.
[0012] In another embodiment, provided herein is a composition
comprising a label for cardiac troponin comprising a detection
binding partner for cardiac troponin I, wherein the detection
binding partner is capable of cross-reacting with cardiac troponin
I from at least two species, and a fluorescent moiety, wherein said
moiety is capable of emitting at least about 200 photons when
simulated by a laser emitting light at the excitation wavelength of
the moiety, wherein the laser is focused on a spot not less than
about 5 microns in diameter that contains the moiety, and wherein
the total energy directed at the spot by the laser is no more than
about 3 microJoules. In some embodiments, the detection binding
partner is capable of reacting with cardiac troponin I from at
least two species selected from the group consisting of human,
monkey, dog, and rat. In some embodiments, the detection binding
partner is capable of reacting with cardiac troponin I from human,
monkey, dog, and rat. The detection binding partner can be an
antibody. In some embodiments, the composition further comprises a
capture binding partner for cardiac troponin I, wherein the capture
binding partner is capable of cross-reacting with cardiac troponin
I from at least two species. The capture binding partner can be
capable of reacting with cardiac troponin I from at least two
species selected from the group consisting of human, monkey, dog,
and rat. In some embodiments, the capture binding partner is
capable of reacting with cardiac troponin I from human, monkey,
dog, and rat. In some embodiments the capture binding partner is an
antibody.
[0013] In some embodiments the invention involves a kit containing
a composition including an antibody to cardiac troponin attached to
a fluorescent dye moiety, where the moiety is capable of emitting
at least about 200 photons when simulated by a laser emitting light
at the excitation wavelength of the moiety, where the laser is
focused on a spot not less than about 5 microns in diameter that
contains the moiety, and where the total energy directed at the
spot by the laser is no more than about 3 microJoules, where the
composition is packaged in suitable packaging. In some embodiments
of the kits of the invention, the cardiac troponin is cardiac
troponin I or cardiac troponin T. In some embodiments, the cardiac
troponin is cardiac troponin I. In some embodiments of the kits of
the invention, the kits further include instructions. In some
embodiments of the kits of the invention, the kits further include
a composition containing a capture antibody for the cardiac
troponin I attached to a solid support. In some embodiments, the
solid support comprises a microtiter plate or paramagnetic
microparticles. In some embodiments of the kits of the invention,
the kits further include a component selected from the group
consisting of wash buffer, assay buffer, elution buffer, and
calibrator diluent. In some embodiments of the kits of the
invention, further include a standard for the cardiac troponin.
INCORPORATION BY REFERENCE
[0014] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0016] FIGS. 1A and 1B. Schematic diagram of the arrangement of the
components of a single particle analyzer. FIG. 1A shows an analyzer
that includes one electromagnetic source and one electromagnetic
detector; FIG. 1B shows an analyzer that includes two
electromagnetic sources and one electromagnetic detectors.
[0017] FIGS. 2A and 2B. Schematic diagrams of a capillary flow cell
for a single particle analyzer. FIG. 2A shows the flow cell of an
analyzer that includes one electromagnetic source; and FIG. 2B
shows the flow cell of an analyzer that includes two
electromagnetic sources.
[0018] FIGS. 3A and 3B. Schematic diagrams showing the conventional
(A) and confocal (B) positioning of laser and detector optics of a
single particle analyzer. FIG. 3A shows the arrangement for an
analyzer that has one electromagnetic source and one
electromagnetic detector; FIG. 3B shows the arrangement for an
analyzer that has two electromagnetic sources and two
electromagnetic detectors.
[0019] FIG. 4. Linearized standard curve for the range
concentrations of cTnI.
[0020] FIG. 5. Biological threshold (cutoff concentration) for cTnI
is at a cTnI concentration of 7 pg/ml, as established at the 99th
percentile with a corresponding CV of 10%.
[0021] FIG. 6. Correlation of assay results of cTnI determined
using the analyzer system of the invention with standard
measurements provided by the National Institute of Standards and
Technology (R2=0.9999).
[0022] FIG. 7. Detection of cTnI in serial serum samples from
patients who presented at the emergency room with chest pain. The
measurements made with the analyzer system of the invention were
compared to measurements made with a commercially available
assay.
[0023] FIG. 8. Distribution of normal biological concentrations of
cTnI (No Ischemia) and concentrations of cTnI in serum samples from
patients presenting with chest pain.
[0024] FIG. 9. Table showing cross-reactivity of capture/detection
antibody pair across human, monkey, dog, and rat.
[0025] FIG. 10. Table showing endogenous serum levels of cTnI in
human, monkey, dog, and rat.
[0026] FIG. 11. Linear regression analysis of back interpolated
values from a typical human IL-6 standard Curve
[0027] FIG. 12. Graph showing interassay precision; back
interpolation of cTnI standard curves generated over 8 consecutive
assay runs. FIG. 12A shows the full range of values; FIG. 12B shows
the low end range of quantification.
[0028] FIG. 13. Frequency distribution of cTnI in lithium heparin
plasma specimens obtained from 100 different blood donors.
[0029] FIGS. 14A and 14B. Distribution of results of biological
variation for cardiac troponin. FIG. 14A shows the short-term
biological variation for cardiac troponin; FIG. 14B shows the
long-term biological variation for cardiac troponin.
[0030] FIG. 15. Graph showing concentrations of cTnI (ng/L) in
matched serum and plasma specimens obtained from 20 human donors of
mixed age and sex. Samples were run in triplicate and results for
each sample are presented as the mean +/- standard deviation.
DETAILED DESCRIPTION OF THE INVENTION
Summary
I. Introduction
II. Cardiac Troponin
III. Labels for Cardiac Troponin
[0031] A. Binding partners for troponin [0032] 1. Antibodies [0033]
2. Cross-reacting antibodies
[0034] B. Fluorescent Moieties To Be Used With Binding Partners
[0035] 1. Dyes [0036] 2. Quantum dots
[0037] C. Binding Partner-Fluorescent Moiety Compositions
IV. Highly Sensitive Analysis of Cardiac Troponin
[0038] A. Sample
[0039] B. Sample preparation
[0040] C. Detection of troponin and determination of
concentration
V. Instruments and Systems Suitable for Highly Sensitive Analysis
of Troponin
[0041] A. Apparatus/System
[0042] B. Single Particle Analyzer [0043] 1. Electromagnetic
Radiation Source [0044] 2. Capillary Flow Cell [0045] 3. Motive
Force [0046] 4. Detectors
[0047] C. Sampling System
[0048] D. Sample preparation system
[0049] E. Sample recovery
VI. Methods Using Highly Sensitive Analysis of Cardiac Troponin
[0050] A. Samples
[0051] B. Determination of diagnosis, prognosis, or method of
treatment [0052] 1. Acute myocardial infarct [0053] 2. Conditions
other than AMI [0054] a. Cardiac toxicity
[0055] C. Business Methods
VII. Compositions
VIII. Kits
I. Introduction
[0056] The invention provides compositions and methods for the
highly sensitive detection of troponin, e.g., cardiac troponin. The
release into the blood of the cardiac isoforms of troponin, which
are unique to cardiac muscle (cardiac troponin I and/or T) is
indicative of damage to cardiac muscle, and provides the basis for
their use as diagnostic or prognostic markers, or to aid in
determination of treatment.
[0057] The troponin complex in muscle consists of troponin I, C and
T. Troponin C exists as two isoforms, one from cardiac and
slow-twitch muscle and one from fast-twitch muscle; because it is
found in virtually all striated muscle, its use as a specific
marker is limited. In contrast, troponin I and T are expressed as
different isoforms in slow-twitch, fast-twitch and cardiac muscle.
The unique cardiac isoforms of troponin I and T allow them to be
distinguished immunologically from the other troponins of skeletal
muscle. Therefore, the release into the blood of cardiac troponin I
and T is indicative of damage to cardiac muscle, and provides the
basis for their use as diagnostic or prognostic markers, or to aid
in determination of treatment.
[0058] Currently used markers for cardiac damage suffer
disadvantages that limit their clinical usefulness. Cardiac enzyme
assays have formed the basis for determining whether or not there
is damage to the cardiac muscle. Unfortunately, the standard
creatine kinase-MB (CK-MB) assay is not reliable in excluding
infarction until 10 to 12 hours after the onset of chest pain.
Earlier diagnosis would have very specific advantages with regard
to fibrinolytic therapy and triage.
[0059] Because the level of troponin found in the circulation of
healthy individuals is very low, and cardiac specific troponins do
not arise from extra-cardiac sources, the troponins are very
sensitive and specific markers of cardiac injury. In addition to
cardiac infarct, a number of other conditions can cause damage to
the heart muscle, and early detection of such damage would prove
useful to clinicians. However, present methods of detection and
quantitation of cardiac troponin do not possess sufficient
sensitivity to detect the release of cardiac troponin into the
blood until levels have reached abnormally high concentrations,
e.g., 0.1 ng/ml or greater.
[0060] The methods and compositions of the invention thus include
methods and compositions for the highly sensitive detection and
quantitation of cardiac troponin, and compositions and methods for
diagnosis, prognosis, and/or determination of treatment based on
such highly sensitive detection and quantitation.
II. Cardiac Troponin
[0061] When the two unique forms of cardiac troponin, cardiac
troponin I (cTnI) and cardiac troponin (cTnT) are released into the
blood from cardiac muscle, several species of each may exist in the
blood. These include various complexes of the two forms, with each
other and/or with cardiac troponin C (cTnC). In addition, the two
forms are subject to virtually immediate proteolytic degradation,
resulting in a variety of fragments. Also, various phosphorylated
and oxidized forms of the troponins may exist in the blood. See,
e.g., U.S. Pat. No. 6,991,907, incorporated by reference herein in
its entirety. Unless otherwise specified, "cardiac troponin," as
used herein, encompasses all forms of cardiac troponin,
including
[0062] In some embodiments, the invention provides methods and
compositions for the detection and/or determination of
concentration of total cardiac troponin, i.e., the sum of all or a
substantial portion of the cardiac troponin in a sample, e.g.,
blood, serum or plasma sample, whether it is free, complexed, a
proteotlytic fragment, phosphorylated, oxidized, or otherwise
modified. In some embodiments, the cardiac troponin is cTnI, in
others, it is cTnT, and in still other embodiments, the cardiac
troponin is cTnI and cTnT. It will be appreciated that an absolute
total measurement need not be achieved, as long as a consistent
proportion of the total is determined, which can be compared to
standard values. It will also be appreciated that if a form of
troponin is a minor constituent of the total, absence or low levels
of detection of that form will not appreciably affect measures of
total troponin. Thus, as used herein, "total cardiac troponin"
refers to a measurement that is intended to measure all or
substantially all forms of a particular cardiac troponin, e.g., all
cTnI, or all cTnT, in a sample, where the sample-to-sample
consistency is such that clinically relevant conclusions may be
drawn from comparisons of samples to standards, or comparison of
one sample to another.
[0063] In some embodiments, the invention provides methods and
compositions for the detection and/or determination of
concentration of one or more of the various forms of troponin in
the sample as a separate entity, e.g., complexed cTnI, free cTnI,
muddied cTnI (e.g., oxidized or phosphorylated), or complexed cTnT,
free cTnT, muddied cTnT (e.g., oxidized or phosphorylated), and,
typically, can provide a concentration for that form in the sample.
In the latter embodiments, ratios or absolute values may be
determined for the different entities. Thus, in some embodiments,
the invention provides methods of detecting and, typically,
determining the concentration of, one or more forms of complexed
troponin, or one or more fragments of troponin, or one or more
oxidized or phosphorylated forms of troponin. In some embodiments,
more than one form is detected, and the concentrations of the
various forms may be determined e.g., by performing multiplexed
assays on a single sample for the different entities, or by
performing separate assays on aliquots from the same or similar
samples. Ratios of concentrations of the various forms may be
obtained. For example, a ratio of the concentration of a particular
form, e.g., a fragment, complex, or modified form, of the cardiac
troponin to the concentration of total cardiac troponin, may be
determined. These ratios and/or absolute values can provide
meaningful clinical information. For example the relative
proportion of fragments of cardiac troponin can indicate the length
of time since release into the blood and thus, indirectly, length
of time since, e.g., myocardial infarct. See, e.g., U.S. Pat. No.
6,991,907, incorporated by reference herein in its entirety.
III. Labels for Cardiac Troponin
[0064] In some embodiments, the invention provides methods and
compositions that include labels for the highly sensitive detection
and quantitation of cardiac troponin.
[0065] One skilled in the art will recognize that many strategies
can be used for labeling target molecules to enable their detection
or discrimination in a mixture of particles. The labels may be
attached by any known means, including methods that utilize
non-specific or specific interactions of label and target. Labels
may provide a detectable signal or affect the mobility of the
particle in an electric field. In addition, labeling can be
accomplished directly or through binding partners.
[0066] In some embodiments, the label comprises a binding partner
to troponin attached to a fluorescent moiety.
[0067] A. Binding Partners for Troponin
[0068] Any suitable binding partner with the requisite specificity
for the form of cardiac troponin to be detected may be used. For
example, a binding partner specific to all or substantially all
forms of cTnI may be used or a binding partner specific to all or
substantially all forms of cTnT may be used; typically such binding
partners bind to a region of the cardiac troponin that is common to
all or most of the different forms likely to be found in a sample.
In some embodiments, a binding partner specific to one or more
particular forms of cardiac troponin may be used, e.g., a binding
partner to complexed cTnI, free cTnI, muddied cTnI (e.g., oxidized
or phosphorylated), or complexed cTnT, free cTnT, muddied cTnT
(e.g., oxidized or phosphorylated). Binding partners are known in
the art and include, e.g., aptamers, lectins, and receptors. A
useful and versatile type of binding partner is an antibody.
[0069] 1. Antibodies
[0070] In some embodiments, the binding partner is an antibody
specific for a cardiac troponin. The term "antibody," as used
herein, is a broad term and is used in its ordinary sense,
including, without limitation, to refer to naturally occurring
antibodies as well as non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. In some embodiments, the antibody is specific
for cTnI. In some embodiments, the antibody is specific for cTnT.
In some embodiments, the label includes antibodies to both cTnI and
cTnT. The antibody may be specific to all or substantially all
forms the cardiac troponin; e.g., all or substantially all forms of
cTnI, or all or substantially all forms of cTnT. In some
embodiments, an antibody specific to one or more particular forms
of cardiac troponin may be used, e.g., a binding partner to
complexed cTnI, free cTnI, muddied cTnI (e.g., oxidized or
phosphorylated), or complexed cTnT, free cTnT, muddied cTnT (e.g.,
oxidized or phosphorylated). Mixtures of antibodies are also
encompassed by the invention, e.g., mixtures of antibodies to cTnI
and cTnT, or mixtures of antibodies to the various forms of the
troponin (free, complexed, etc.), or mixtures of mixtures.
[0071] It will be appreciated that the choice of epitope or region
of troponin to which the antibody is raised will determine its
specificity, e.g., for total troponin, for certain fragments, for
complexed troponin, for modified troponin, and the like. In some
embodiments, the antibody is specific to a specific amino acid
region of a cardiac troponin. In some embodiments, the antibody is
specific to amino acids 27-41 of human cardiac troponin I. Both
monoclonal and polyclonal antibodies are useful as binding
partners. In some embodiments, the antibody is a polyclonal
antibody. In some embodiments, the antibody is a monoclonal
antibody. In some embodiments, the antibody is a polyclonal
antibody specific to amino acids 27-41 of human cardiac troponin I.
In some embodiments, this antibody is not affected by heparin,
phosphorylation, oxidation and troponin complex formation, and does
not cross-react with skeletal muscle troponin I.
[0072] Methods for producing antibodies are well-established. The
cardiac specific sequences to troponin I and troponin T are
described in FEBS Lett. 270, 57-61 (1990) and Genomics 21, 311-316
(1994). One skilled in the art will recognize that many procedures
are available for the production of antibodies, for example, as
described in Antibodies, A Laboratory Manual, Ed Harlow and David
Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor,
N.Y. One skilled in the art will also appreciate that binding
fragments or Fab fragments which mimic antibodies can also be
prepared from genetic information by various procedures (Antibody
Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995,
Oxford University Press, Oxford; J. Immunol. 149, 3914-3920
(1992)). Methods for raising antibodies to the various complexed,
fragment, phosphorylated, and oxidized forms of the troponins are
disclosed in U.S. Pat. Nos. 5,579,687; 6,991,907; and in US Patent
Application No. 20050164317, which are herein incorporated by
reference in their entirety. A synthetic peptide comprised of 14
amino acids which mimics a cardiac specific sequence of troponin I
and methods used to prepare antibodies to the peptide are described
in international Patent Application number PCT/US94/05468.
Monoclonal and polyclonal antibodies to free and complexed cardiac
troponins are also commercially available (HyTest, HyTest Ltd.,
Turku Finland; Abcam Inc., Cambridge, Mass., USA, Life Diagnostics,
Inc., West Chester, Pa., USA; Fitzgerald Industries International,
Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville,
Calif.).
[0073] In some embodiments, the antibody is a mammalian, e.g., goat
polyclonal anti-cTnI antibody. The antibody may be specific to
specific regions of the cTnI, e.g., amino acids 27-41 of human
cardiac troponin I. Capture binding partners and detection binding
partner pairs, e.g., capture and detection antibody pairs, may be
used in embodiments of the invention. Thus, in some embodiments, a
heterogeneous assay protocol is used in which, typically, two
binding partners, e.g., two antibodies, are used. One binding
partner is a capture partner, usually immobilized on a solid
support, and the other binding partner is a detection binding
partner, typically with a detectable label attached. In some
embodiments, the capture binding partner member of a pair is an
antibody that is specific to all or substantially all forms of
cardiac troponin. An example is an antibody, e.g., a monoclonal
antibody, specific to free cardiac troponin I (cTnI) a.a. 41-49 and
cTnI forming complexes with other troponin components. Preferably,
this antibody is not affected by heparin, phosphorylation,
oxidation and troponin complex formation, and does not cross-react
with skeletal muscle troponin I. Thus, it is thought that the
antibody binds to total cTnI. Another example is a monoclonal
antibody, specific to cardiac troponin I (cTnI) a.a. 87-91 and does
not cross-react with skeletal muscle troponin I. Such antibodies
are available from BiosPacific, Emeryville, Calif. Other antibody
pairs are known or can be designed.
[0074] Cross-reacting antibodies In some embodiments it is useful
to use an antibody that cross-reacts with a variety of species,
either as a capture antibody, a detection antibody, or both. Such
embodiments include the measurement of drug toxicity by
determining, e.g., release of cardiac troponin into the blood as a
marker of cardiac damage. A cross-reacting antibody allows studies
of toxicity to be done in one species, e.g. a non-human species,
and direct transfer of the results to studies or clinical
observations of another species, e.g., humans, using the same
antibody or antibody pair in the reagents of the assays, thus
decreasing variability between assays. Thus, in some embodiments,
one or more of the antibodies for use as a binding partner to the
marker, e.g., cardiac troponin, such as cardiac troponin I, may be
a cross-reacting antibody. In some embodiments, the antibody
cross-reacts with the marker, e.g. cardiac troponin, from at least
two species selected from the group consisting of human, monkey,
dog, rat, and mouse. In some embodiments the antibody cross-reacts
with the marker e.g. cardiac troponin, from all of the group
consisting of human, monkey, dog, rat, and mouse. In some
embodiments, the antibody cross-reacts with cardiac troponin, e.g.,
cardiac troponin I, from human, monkey, dog, and rat. Such a
cross-reacting antibody may be used in any suitable embodiment as
described herein. For example, a cross-reacting antibody pair may
be used wherein each member of the pair (i.e., capture and
detection antibody) cross reacts across species with cardiac
troponin I. The capture antibody may be immobilized on a solid
support. In some embodiments, the capture antibody is immobilized
on a paramagnetic microparticle, such as described in Example 5. An
example of results obtained from a cross-reacting antibody pair for
human, monkey, dog, and rat is given in Example 6.
[0075] B. Fluorescent Moieties to be Used with Binding Partners
[0076] In some embodiments, the binding partner, e.g., antibody, is
attached to a fluorescent moiety. The fluorescence of the moiety,
will be sufficient to allow detection in a single molecule
detector, such as the single molecule detectors described herein. A
"fluorescent moiety," as that term is used herein, includes one or
more fluorescent entities whose total fluorescence is such that the
moiety may be detected in the single molecule detectors described
herein. Thus, a fluorescent moiety may comprise a single entity
(e.g., a Quantum Dot or fluorescent molecule) or a plurality of
entities (e.g., a plurality of fluorescent molecules). It will be
appreciated that when "moiety," as that term is used herein, refers
to a group of fluorescent entities, e.g., a plurality of
fluorescent dye molecules, each individual entity may be attached
to the binding partner separately or the entities may be attached
together, as long as the entities as a group provide sufficient
fluorescence to be detected.
[0077] Typically, the fluorescence of the moiety involves a
combination of quantum efficiency and lack of photobleaching
sufficient that the moiety is detectable above background levels in
a single molecule detector, with the consistency necessary for the
desired level of detection, accuracy, and precision of the assay.
For example, in some embodiments, the fluorescence of the
fluorescent moiety is such that it allows detection and/or
quantitation of troponin at a level of detection of less than about
10, 5, 4, 3, 2, or 1 pg/ml and with a coefficient of variation of
less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
1% or less, e.g., about 10% or less, in the instruments described
herein. In some embodiments, the fluorescence of the fluorescent
moiety is such that it allows detection and/or quantitation of
troponin at a limit of detection of less than about 5 pg/ml and
with a coefficient of variation of less than about 10%, in the
instruments described herein. "Limit of detection," as that term is
used herein, includes the lowest concentration at which one can
identify a sample as containing a molecule of the substance of
interest, e.g., the first non-zero value. It can be defined by the
variability of zeros and the slope of the standard curve. For
example, the limit of detection of an assay may be determined by
running a standard curve, determining the standard curve zero
value, and adding 2 standard deviations to that value. A
concentration of the substance of interest that produces a signal
equal to this value is the "lower limit of detection"
concentration.
[0078] Furthermore, the moiety has properties that are consistent
with its use in the assay of choice. In some embodiments, the assay
is an immunoassay, where the fluorescent moiety is attached to an
antibody; the moiety must have properties such that it does not
aggregate with other antibodies or proteins, or experiences no more
aggregation than is consistent with the required accuracy and
precision of the assay. In some embodiments, fluorescent moieties
that are preferred are fluorescent moieties, e.g., dye molecules
that have a combination of 1) high absorption coefficient; 2) high
quantum yield; 3) high photostability (low photobleaching); and 4)
compatibility with labeling the biomolecule of interest (e.g.,
protein) so that it may be analyzed using the analyzers and systems
of the invention (e.g., does not cause precipitation of the protein
of interest, or precipitation of a protein to which the moiety has
been attached).
[0079] Fluorescent moieties, e.g. a single fluorescent dye molecule
or a plurality of fluorescent dye molecules, that are useful in
some embodiments of the invention may be defined in terms of their
photon emission characteristics when stimulated by EM radiation.
For example, in some embodiments, the invention utilizes a
fluorescent dye moiety, e.g., a single fluorescent dye molecule or
a plurality of fluorescent dye molecules, that is capable of
emitting an average of at least about 10, 20, 30, 40, 50, 75, 100,
125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700,
800, 900, or 1000, photons when simulated by a laser emitting light
at the excitation wavelength of the moiety, where the laser is
focused on a spot of not less than about 5 microns in diameter that
contains the moiety, and wherein the total energy directed at the
spot by the laser is no more than about 3 microJoules. It will be
appreciated that the total energy may be achieved by many different
combinations of power output of the laser and length of time of
exposure of the dye moiety. E.g., a laser of a power output of 1 mW
may be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for
0.25 ms, and so on.
[0080] In some embodiments, the invention utilizes a fluorescent
dye moiety, e.g., a single fluorescent dye molecule or a plurality
of fluorescent dye molecules, that is capable of emitting an
average of at least about 50 photons when simulated by a laser
emitting light at the excitation wavelength of the moiety, where
the laser is focused on a spot of not less than about 5 microns in
diameter that contains the moiety, and wherein the total energy
directed at the spot by the laser is no more than about 3
microJoules. In some embodiments, the invention utilizes a
fluorescent dye moiety, e.g., a single fluorescent dye molecule or
a plurality of fluorescent dye molecules, that is capable of
emitting an average of at least about 100 photons when simulated by
a laser emitting light at the excitation wavelength of the moiety,
where the laser is focused on a spot of not less than about 5
microns in diameter that contains the moiety, and wherein the total
energy directed at the spot by the laser is no more than about 3
microJoules. In some embodiments, the invention utilizes a
fluorescent dye moiety, e.g., a single fluorescent dye molecule or
a plurality of fluorescent dye molecules, that is capable of
emitting an average of at least about 150 photons when simulated by
a laser emitting light at the excitation wavelength of the moiety,
where the laser is focused on a spot of not less than about 5
microns in diameter that contains the moiety, and wherein the total
energy directed at the spot by the laser is no more than about 3
microJoules. In some embodiments, the invention utilizes a
fluorescent dye moiety, e.g., a single fluorescent dye molecule or
a plurality of fluorescent dye molecules, that is capable of
emitting an average of at least about 200 photons when simulated by
a laser emitting light at the excitation wavelength of the moiety,
where the laser is focused on a spot of not less than about 5
microns in diameter that contains the moiety, and wherein the total
energy directed at the spot by the laser is no more than about 3
microJoules. In some embodiments, the invention utilizes a
fluorescent dye moiety, e.g., a single fluorescent dye molecule or
a plurality of fluorescent dye molecules, that is capable of
emitting an average of at least about 300 photons when simulated by
a laser emitting light at the excitation wavelength of the moiety,
where the laser is focused on a spot of not less than about 5
microns in diameter that contains the moiety, and wherein the total
energy directed at the spot by the laser is no more than about 3
microJoules. In some embodiments, the invention utilizes a
fluorescent dye moiety e.g., a single fluorescent dye molecule or a
plurality of fluorescent dye molecules, that is capable of emitting
an average of at least about 500 photons when simulated by a laser
emitting light at the excitation wavelength of the moiety, where
the laser is focused on a spot of not less than about 5 microns in
diameter that contains the moiety, and wherein the total energy
directed at the spot by the laser is no more than about 3
microJoules.
[0081] In some embodiments, the fluorescent moiety comprises an
average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or fluorescent
entities, e.g., fluorescent molecules. In some embodiments, the
fluorescent moiety comprises an average of no more than about 2, 3,
4, 5, 6, 7, 8, 9, 10, or 11 fluorescent entities, e.g., fluorescent
molecules. In some embodiments, the fluorescent moiety comprises an
average of about 1 to 11, or about 2 to 10, or about 2 to 8, or
about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10,
or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to
10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or
more than about 6 fluorescent entities. In some embodiments, the
fluorescent moiety comprises an average of about 2 to 8 fluorescent
moieties are attached. In some embodiments, an average of about 2
to 6 fluorescent entities. In some embodiments, the fluorescent
moiety comprises an average of about 2 to 4 fluorescent entities.
In some embodiments, the fluorescent moiety comprises an average of
about 3 to 10 fluorescent entities. In some embodiments, the
fluorescent moiety comprises an average of about 3 to 8 fluorescent
entities. In some embodiments, the fluorescent moiety comprises an
average of about 3 to 6 fluorescent entities. By "average" is meant
that, in a given sample that is a representative sample of a group
of labels of the invention, where the sample contains a plurality
of the binding partner-fluorescent moiety units, the molar ratio of
the particular fluorescent entity of which the fluorescent moiety
is comprise, to the binding partner, as determined by standard
analytical methods, corresponds to the number or range of numbers
specified For example, in embodiments in which the label comprises
a binding partner that is an antibody and a fluorescent moiety that
comprises a plurality of fluorescent dye molecules of a specific
absorbance, a spectrophometric assay may be used in which a
solution of the label is diluted to an appropriate level and the
absorbance at 280 nm is taken to determine the molarity of the
protein (antibody) and an absorbance at, e.g., 650 nm (for
AlexaFluor 647) is taken to determine the molarity of the
fluorescent dye molecule. The ratio of the latter molarity to the
former represents the average number of fluorescent entities (dye
molecules) in the fluorescent moiety attached to each antibody.
[0082] 1. Dyes
[0083] In some embodiments, the invention utilizes fluorescent
moieties that comprise fluorescent dye molecules. In some
embodiments, the invention utilizes a fluorescent dye molecule that
is capable of emitting an average of at least about 50 photons when
simulated by a laser emitting light at the excitation wavelength of
the molecule, where the laser is focused on a spot of not less than
about 5 microns in diameter that contains the molecule, and wherein
the total energy directed at the spot by the laser is no more than
about 3 microJoules. In some embodiments, the invention utilizes a
fluorescent dye molecule that is capable of emitting an average of
at least about 75 photons when simulated by a laser emitting light
at the excitation wavelength of the molecule, where the laser is
focused on a spot of not less than about 5 microns in diameter that
contains the molecule, and wherein the total energy directed at the
spot by the laser is no more than about 3 microJoules. In some
embodiments, the invention utilizes a fluorescent dye molecule that
is capable of emitting an average of at least about 100 photons
when simulated by a laser emitting light at the excitation
wavelength of the molecule, where the laser is focused on a spot of
not less than about 5 microns in diameter that contains the
molecule, and wherein the total energy directed at the spot by the
laser is no more than about 3 microJoules. In some embodiments, the
invention utilizes a fluorescent dye molecule that is capable of
emitting an average of at least about 150 photons when simulated by
a laser emitting light at the excitation wavelength of the
molecule, where the laser is focused on a spot of not less than
about 5 microns in diameter that contains the molecule, and wherein
the total energy directed at the spot by the laser is no more than
about 3 microJoules. In some embodiments, the invention utilizes a
fluorescent dye molecule that is capable of emitting an average of
at least about 200 photons when simulated by a laser emitting light
at the excitation wavelength of the molecule, where the laser is
focused on a spot of not less than about 5 microns in diameter that
contains the molecule, and wherein the total energy directed at the
spot by the laser is no more than about 3 microJoules
[0084] A non-inclusive list of useful fluorescent entities for use
in the fluorescent moieties of the invention is given in Table 1,
below. In some embodiments, the fluorescent entity is selected from
the group consisting of Alexa Flour 488, 532, 647, 700, 750,
Fluorescein, B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot
605.
TABLE-US-00001 TABLE 1 FLUORESCENT ENTITIES Em Dye Ex (nm) E (M) -
1 (nm) Mw Bimane 380 5,700 458 282.31 Dapoxyl 373 22,000 551 362.83
Dimethylamino 375 22,000 470 344.32 coumarin-4-acetic acid Marina
blue 365 19,000 460 367.26 8-Anilino 372 480 naphthalene-1-sulfonic
acid Cascade blue 376 23,000 420 607.42 Alexa Fluor 405 402 35,000
421 1028.26 Cascade blue 400 29,000 420 607.42 Cascade yellow 402
24,000 545 563.54 Pacific blue 410 46,000 455 339.21 PyMPO 415
26,000 570 582.41 Alexa 430 433 15,000 539 701.75 Atto-425 438 486
NBD 465 22,000 535 391.34 Alexa 488 495 73,000 519 643.41
Fluorescein 494 79,000 518 376.32 Oregon Green 488 496 76,000 524
509.38 Atto 495 495 522 Cy2 489 150,000 506 713.78 DY-480-XL 500
40,000 630 514.60 DY-485-XL 485 20,000 560 502.59 DY-490-XL 486
27,000 532 536.58 DY-500-XL 505 90,000 555 596.68 DY-520-XL 520
40,000 664 514.60 Alexa Fluor 532 531 81,000 554 723.77 BODIPY
530/550 534 77,000 554 513.31 6-HEX 535 98,000 556 680.07 6-JOE 522
75,000 550 602.34 Rhodamine 6G 525 108,000 555 555.59 Atto-520 520
542 Cy3B 558 130,000 572 658.00 Alexa Fluor 610 612 138,000 628
Alexa Fluor 633 632 159,000 647 ca. 1200 Alexa Fluor 647 650
250,000 668 ca. 1250 BODIPY 630/650 625 101,000 640 660.50 Cy5 649
250,000 670 791.99 Alexa Fluor 660 663 110,000 690 Alexa Fluor 680
679 184,000 702 Alexa Fluor 700 702 192,000 723 Alexa Fluor 750 749
240,000 782 B-phycoerythrin 546, 2,410,000 575 240,000 565
R-phycoerythrin 480, 1,960,000 578 240,000 546, 565 Allophycocyanin
650 700,000 660 700,000 PBXL-1 545 666 PBXL-3 614 662 Atto-tec dyes
Name Ex (nm) Em (nm) QY .quadrature. (ns) Atto 425 436 486 0.9 3.5
Atto 495 495 522 0.45 2.4 Atto 520 520 542 0.9 3.6 Atto 560 561 585
0.92 3.4 Atto 590 598 634 0.8 3.7 Atto 610 605 630 0.7 3.3 Atto 655
665 690 0.3 1.9 Atto 680 680 702 0.3 1.8
Dyomics Fluors
TABLE-US-00002 [0085] Molar molecular absorbance* weight# label Ex
(nm) [l mol-1 cm-1] Em (nm) [g mol-1] DY-495/5 495 70,000 520
489.47 DY-495/6 495 70,000 520 489.47 DY-495X/5 495 70,000 520
525.95 DY-495X/6 495 70,000 520 525.95 DY-505/5 505 85,000 530
485.49 DY-505/6 505 85,000 530 485.49 DY-505X/5 505 85,000 530
523.97 DY-505X/6 505 85,000 530 523.97 DY-550 553 122,000 578
667.76 DY-555 555 100.000 580 636.18 DY-610 609 81.000 629 667.75
DY-615 621 200.000 641 578.73 DY-630 636 200.000 657 634.84 DY-631
637 185.000 658 736.88 DY-633 637 180.000 657 751.92 DY-635 647
175.000 671 658.86 DY-636 645 190.000 671 760.91 DY-650 653 170.000
674 686.92 DY-651 653 160.000 678 888.96 DYQ-660 660 117,000 --
668.86 DYQ-661 661 116,000 -- 770.90 DY-675 674 110.000 699 706.91
DY-676 674 145.000 699 807.95 DY-680 690 125.000 709 634.84 DY-681
691 125.000 708 736.88 DY-700 702 96.000 723 668.86 DY-701 706
115.000 731 770.90 DY-730 734 185.000 750 660.88 DY-731 736 225.000
759 762.92 DY-750 747 240.000 776 712.96 DY-751 751 220.000 779
814.99 DY-776 771 147.000 801 834.98 DY-780-OH 770 70.000 810
757.34 DY-780-P 770 70.000 810 957.55 DY-781 783 98.000 800 762.92
DY-782 782 102.000 800 660.88 EVOblue-10 651 101.440 664 389.88
EVOblue-30 652 102.000 672 447.51
Quantum Dots: Qdot 525, 565, 585, 605, 655, 705, 800
[0086] Suitable dyes for use in the invention include modified
carbocyanine dyes. The modification of carbocyanine dyes includes
the modification of an indolium ring of the carbocyanine dye to
permit a reactive group or conjugated substance at the number 3
position. The modification of the indolium ring provides dye
conjugates that are uniformly and substantially more fluorescent on
proteins, nucleic acids and other biopolymers, than conjugates
labeled with structurally similar carbocyanine dyes bound through
the nitrogen atom at the number one position. In addition to having
more intense fluorescence emission than structurally similar dyes
at virtually identical wavelengths, and decreased artifacts in
their absorption spectra upon conjugation to biopolymers, the
modified carbocyanine dyes have greater photostability and higher
absorbance (extinction coefficients) at the wavelengths of peak
absorbance than the structurally similar dyes. Thus, the modified
carbocyanine dyes result in greater sensitivity in assays that use
the modified dyes and their conjugates. Preferred modified dyes
include compounds that have at least one substituted indolium ring
system in which the substituent on the 3-carbon of the indolium
ring contains a chemically reactive group or a conjugated
substance. Other dye compounds include compounds that incorporate
an azabenzazolium ring moiety and at least one sulfonate moiety.
The modified carbocyanine dyes that can be used to detect
individual particles in various embodiments of the invention are
described in U.S. Pat. No. 6,977,305, which is herein incorporated
by reference in its entirety. Thus, in some embodiments the labels
of the invention utilize a fluorescent dye that includes a
substituted indolium ring system in which the substituent on the
3-carbon of the indolium ring contains a chemically reactive group
or a conjugated substance group.
[0087] In some embodiments, the label comprises a fluorescent
moiety that includes one or more Alexa dyes (Molecular Probes,
Eugene, Oreg.). The Alexa dyes are disclosed in U.S. Pat. Nos.
6,977,305; 6,974,874; 6,130,101; and 6,974,305, which are herein
incorporated by reference in their entirety. Some embodiments of
the invention utilize a dye chosen from the group consisting of
AlexaFluor 647, AlexaFluor 488, AlexaFluor 532, AlexaFluor 555,
AlexaFluor 610, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750.
Some embodiments of the invention utilize a dye chosen from the
group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647,
AlexaFluor 700 and AlexaFluor 750. Some embodiments of the
invention utilize the AlexaFluor 647 molecule, which has an
absorption maximum between about 650 and 660 nm and an emission
maximum between about 660 and 670 nm. The AlexaFluor 647 dye is
used alone or in combination with other AlexaFluor dyes.
[0088] In addition, currently available organic fluors can be
improved by rendering them less hydrophobic by adding hydrophilic
groups such as polyethylene. Alternatively, currently sulfonated
organic fluors such as the AlexaFluor 647 dye can be rendered less
acidic by making them zwitterionic. Particles such as antibodies
that are labeled with the modified fluors are less likely to bind
non-specifically to surfaces and proteins in immunoassays, and thus
enable assays that have greater sensitivity and lower backgrounds.
Methods for modifying and improving the properties of fluorescent
dyes for the purpose of increasing the sensitivity of a system that
detects single particles are known in the art. Preferably, the
modification improves the Stokes shift while maintaining a high
quantum yield.
[0089] 2 Quantum Dots
[0090] In some embodiments, the fluorescent label moiety that is
used to detect a molecule in a sample using the analyzer systems of
the invention is a quantum dot. Quantum dots (QDs), also known as
semiconductor nanocrystals or artificial atoms, are semiconductor
crystals that contain anywhere between 100 to 1,000 electrons and
range from 2-10 nm. Some QDs can be between 10-20 nm in diameter.
QDs have high quantum yields, which makes them particularly useful
for optical applications. QDs are fluorophores that fluoresce by
forming excitons, which can be thought of the excited state of
traditional fluorophores, but have much longer lifetimes of up to
200 nanoseconds. This property provides QDs with low
photobleaching. The energy level of QDs can be controlled by
changing the size and shape of the QD, and the depth of the QDs'
potential. One of the optical features of small excitonic QDs is
coloration, which is determined by the size of the dot. The larger
the dot, the redder, or more towards the red end of the spectrum
the fluorescence. The smaller the dot, the bluer or more towards
the blue end it is. The bandgap energy that determines the energy
and hence the color of the fluoresced light is inversely
proportional to the square of the size of the QD. Larger QDs have
more energy levels which are more closely spaced, thus allowing the
QD to absorb photons containing less energy, i.e. those closer to
the red end of the spectrum. Because the emission frequency of a
dots dependent on the bandgap, it is therefore possible to control
the output wavelength of a dot with extreme precision. In some
embodiments the protein that is detected with the single particle
analyzer system is labeled with a QD. In some embodiments, the
single particle analyzer is used to detect a protein labeled with
one QD and using a filter to allow for the detection of different
proteins at different wavelengths.
[0091] QDs have broad excitation and narrow emission properties
which when used with color filtering require only a single
electromagnetic source for multiplex analysis of multiple targets
in a single sample to resolve individual signals. Thus, in some
embodiments, the analyzer system comprises one continuous wave
laser and particles that are each labeled with one QD. Colloidally
prepared QDs are free floating and can be attached to a variety of
molecules via metal coordinating functional groups. These groups
include but are not limited to thio, amine, nitrile, phosphine,
phosphine oxide, phosphonic acid, carboxylic acids or other
ligands. By bonding appropriate molecules to the surface, the
quantum dots can be dispersed or dissolved in nearly any solvent or
incorporated into a variety of inorganic and organic films. Quantum
dots (QDs) can be coupled to streptavidin directly through a
maleimide ester coupling reaction or to antibodies through a
meleimide-thiol coupling reaction. This yields a material with a
biomolecule covalently attached on the surface, which produces
conjugates with high specific activity. In some embodiments, the
protein that is detected with the single particle analyzer is
labeled with one quantum dot. In some embodiments the quantum dot
is between 10 and 20 nm in diameter. In other embodiments, the
quantum dot is between 2 and 10 nm in diameter. Useful Quantum Dots
include QD 605, QD 610, QD 655, and QD 705. A particularly
preferred Quantum Dot is QD 605.
[0092] C. Binding Partner-Fluorescent Moiety Compositions
(Labels)
[0093] The labels of the invention generally contain a binding
partner, e.g., antibody, bound to a fluorescent moiety to provide
the requisite fluorescence for detection and quantitation in the
instruments described herein. Any suitable combination of binding
partner and fluorescent moiety for detection in the single molecule
detectors described herein may be used as a label in the invention.
In some embodiments, the invention provides a label for a cardiac
troponin molecule, or fragment, complex, phosphorylated, or
oxidized form thereof, where the label includes an antibody to a
cardiac troponin and a fluorescent moiety. The antibody may be any
antibody as described above, e.g., an antibody to cTnT or cTnI. In
some embodiments, the antibody is an antibody to cTnI. In some
embodiments, the antibody is specific to a specific region of the
cardiac troponin, e.g., specific to amino acids 27-41 of human
cTnI. In some embodiments, the invention provides compositions
comprising a fluorescent moiety attached to an anti-cTnI antibody,
e.g., a polyclonal antibody such as a goat polyclonal antibody from
those designated G129C available from BiosPacific, Emeryville. A
fluorescent moiety may be attached such that the label is capable
of emitting an average of at least about 50, 75, 100, 125, 150,
175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or
1000, photons when simulated by a laser emitting light at the
excitation wavelength of the moiety, where the laser is focused on
a spot of not less than about 5 microns in diameter that contains
the label, and wherein the total energy directed at the spot by the
laser is no more than about 3 microJoules. In some embodiments, the
fluorescent moiety may be a fluorescent moiety that is capable of
emitting an average of at least about 50, 100, 150, or 200 photons
when simulated by a laser emitting light at the excitation
wavelength of the moiety, where the laser is focused on a spot of
not less than about 5 microns in diameter that contains the moiety,
and wherein the total energy directed at the spot by the laser is
no more than about 3 microJoules. The fluorescent moiety may be a
fluorescent moiety that includes one or more dye molecules with a
structure that includes a substituted indolium ring system in which
the substituent on the 3-carbon of the indolium ring contains a
chemically reactive group or a conjugated substance group. The
label composition may include a fluorescent moiety that includes
one or more dye molecules selected from the group consisting of
AlexaFluor 488, 532, 647, 700, or 750. The label composition may
include a fluorescent moiety that includes one or more dye
molecules selected from the group consisting of AlexaFluor 488,
532, 700, or 750. The label composition may include a fluorescent
moiety that includes one or more dye molecules that are AlexaFluor
488. The label composition may include a fluorescent moiety that
includes one or more dye molecules that are AlexaFluor 555. The
label composition may include a fluorescent moiety that includes
one or more dye molecules that are AlexaFluor 610. The label
composition may include a fluorescent moiety that includes one or
more dye molecules that are AlexaFluor 647. The label composition
may include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 680. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 700. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 750.
[0094] In some embodiments the invention provides a composition for
the detection of cardiac troponin I that includes an AlexFluor
molecule, e.g. an AlexaFluor molecule selected from the described
groups, such as an AlexaFluor 647 molecule attached to a to an
antibody, e.g., a goat polyclonal anti-cTnI antibody, specific for
amino acids 27-41 of human cTnI. In some embodiments the invention
provides a composition for the detection of cardiac troponin I that
includes an average of 1 to 11, or about 2 to 10, or about 2 to 8,
or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to
10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4
to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or
more than about 6 AlexaFluor 647 molecules molecule attached an
antibody, e.g., a goat polyclonal anti-cTnI antibody, specific for
amino acids 27-41 of human cTnI. In some embodiments the invention
provides a composition for the detection of cardiac troponin I that
includes an average of 1 to 11, or about 2 to 10, or about 2 to 8,
or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 to
10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4
to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or
more than about 6 AlexaFluor 647 molecules molecule attached to an
antibody, e.g., a goat polyclonal anti-cTnI antibody, specific for
amino acids 27-41 of human cTnI. In some embodiments the invention
provides a composition for the detection of cardiac troponin I that
includes an average of about 2 to 10 AlexaFluor 647 molecules
molecule attached to an antibody, e.g., a goat polyclonal anti-cTnI
antibody, specific for amino acids 27-41 of human cTnI. In some
embodiments the invention provides a composition for the detection
of cardiac troponin I that includes an average of about 2 to 8
AlexaFluor 647 molecules molecule attached to an antibody, e.g., a
goat polyclonal anti-cTnI antibody, specific for amino acids 27-41
of human cTnI. In some embodiments the invention provides a
composition for the detection of cardiac troponin I that includes
an average of about 2 to 6 AlexaFluor 647 molecules molecule
attached to an antibody, e.g., a goat polyclonal anti-cTnI
antibody, specific for amino acids 27-41 of human cTnI. In some
embodiments the invention provides a composition for the detection
of cardiac troponin I that includes an average of about 2 to 4
AlexaFluor 647 molecules molecule attached to an antibody, e.g., a
goat polyclonal anti-cTnI antibody, specific for amino acids 27-41
of human cTnI. In some embodiments the invention provides a
composition for the detection of cardiac troponin I that includes
an average of about 3 to 8 AlexaFluor 647 molecules molecule
attached to an antibody, e.g., a goat polyclonal anti-cTnI
antibody, specific for amino acids 27-41 of human cTnI. In some
embodiments the invention provides a composition for the detection
of cardiac troponin I that includes an average of about 3 to 6
AlexaFluor 647 molecules molecule attached to an antibody, e.g., a
goat polyclonal anti-cTnI antibody, specific for amino acids 27-41
of human cTnI. In some embodiments the invention provides a
composition for the detection of cardiac troponin I that includes
an average of about 4 to 8 AlexaFluor 647 molecules molecule
attached to an antibody, e.g., a goat polyclonal anti-cTnI
antibody, specific for amino acids 27-41 of human cTnI.
[0095] Attachment of the fluorescent moiety, or fluorescent
entities that make up the fluorescent moiety, to the binding
partner, e.g., antibody, may be by any suitable means; such methods
are well-known in the art and exemplary methods are given in the
Examples. In some embodiments, after attachment of the fluorescent
moiety to the binding partner to form a label for use in the
methods of the invention, and prior to the use of the label for
labeling the protein of interest, it is useful to perform a
filtration step. E.g., an antibody-dye label may be filtered prior
to use, e.g., through a 0.2 micron filter, or any suitable filter
for removing aggregates. Other reagents for use in the assays of
the invention may also be filtered, e.g., e.g., through a 0.2
micron filter, or any suitable filter. Without being bound by
theory, it is thought that such filtration removes a portion of the
aggregates of the, e.g., antibody-dye labels. As such aggregates
will bind as a unit to the protein of interest, but upon release in
elution buffer are likely to disaggregate, false positives may
result; i.e., several labels will be detected from an aggregate
that has bound to only a single protein molecule of interest.
Regardless of theory, filtration has been found to reduce false
positives in the subsequent assay and to improve accuracy and
precision.
IV. Highly Sensitive Analysis of Cardiac Troponin
[0096] In one aspect, the invention provides a method for
determining the presence or absence of a single molecule of cardiac
troponin or a fragment or complex thereof in a sample, by i)
labeling the molecule, fragment, or complex, if present, with a
label; and ii) detecting the presence or absence of the label,
where the detection of the presence of the label indicates the
presence of the single molecule, fragment, or complex of cardiac
troponin in the sample. As used herein, "molecule of cardiac
troponin" includes a molecule that contains substantially the
entire naturally-occurring amino acid sequence of the particular
type of cardiac troponin, including post-translationally modified
forms, e.g., phosphorylated forms, as well as oxidized or otherwise
chemically altered forms. As used herein, a "fragment" of a
molecule includes a molecule of cardiac troponin that contains less
than the entire naturally-occurring amino acid sequence, including
modifications as for the entire molecule. As used herein, a
"complex" of a molecule of cardiac troponin includes a molecule of
cardiac troponin or a fragment that is associated with one or more
other molecules or substances, e.g., that is associated with one or
more other molecules of cardiac troponin. In some embodiments, the
method is capable of detecting the troponin at a limit of detection
of less than about 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8,
7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2. or 0.1
pg/ml. In some embodiments, the method is capable of detecting the
troponin at a limit of detection of less than about 100 pg/ml. In
some embodiments, the method is capable of detecting the troponin
at a limit of detection of less than about 50 pg/ml. In some
embodiments, the method is capable of detecting the troponin at a
limit of detection of less than about 20 pg/ml. In some
embodiments, the method is capable of detecting the troponin at a
limit of detection of less than about 10 pg/ml. In some
embodiments, the method is capable of detecting the troponin at a
limit of detection of less than about 5 pg/ml. In some embodiments,
the method is capable of detecting the troponin at a limit of
detection of less than about 3 pg/ml. In some embodiments, the
method is capable of detecting the troponin at a limit of detection
of less than about 1 pg/ml. Detection limits may be determined by
use of the appropriate National Institute of Standards and
Technology reference standard material, e.g., standard cTnI.
[0097] The methods also provide methods of determining a
concentration of cardiac troponin in a sample by detecting single
molecules of troponin in the sample. The "detecting" of a single
molecule of troponin includes detecting the molecule directly or
indirectly. In the case of indirect detection, labels that
corresponds to single molecules of cardiac troponin, e.g., a labels
that have been attached to the single molecules of cardiac
troponin, may be detected.
[0098] Types of cardiac troponin for detection are as described
herein, e.g., cTnT, cTnI, total cardiac troponin (e.g., total cTnI
or total cTnT) or free, complexed, or fragments of cardiac
troponin. In some embodiments, total cardiac troponin is detected
and/or quantitated. In some embodiments, total cTnT is detected. In
some embodiments, total cTnI is detected and/or quantitated.
[0099] The methods also provide for the measurement of low levels
of biomarkers other than troponin. For example, the detection of
cytokines in plasma in the absence of inflammation, at, for
example, the level of cytokines known to exist in the circulating
plasma proteome of a healthy individual (ref. 2). Another
non-limiting example is the detection and measurement of p24, a
viral protein present in HIV infected individuals, which is present
in the early stages of HIV infection well before HIV antibodies are
produced and can be detected (ref. 12).
[0100] A. Sample
[0101] The sample may be any suitable sample. Typically, the sample
is a biological sample, e.g., a biological fluid. Such fluids
include, without limitation, exhaled breath condensate (EBC),
bronchoalveolar lavage fluid (BAL), blood, serum, plasma, urine,
cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal
fluid, amniotic fluid, gastric fluid, lymph fluid, interstitial
fluid, tissue homogenate, cell extracts, saliva, sputum, stool,
physiological secretions, tears, mucus, sweat, milk, semen, seminal
fluid, vaginal secretions, fluid from ulcers and other surface
eruptions, blisters, and abscesses, and extracts of tissues
including biopsies of normal, malignant, and suspect tissues or any
other constituents of the body which may contain the target
particle of interest. Other similar specimens such as cell or
tissue culture or culture broth are also of interest.
[0102] In some embodiments, the sample is a blood sample. In some
embodiments the sample is a plasma sample. In some embodiments the
sample is a serum sample.
[0103] B. Sample Preparation
[0104] In general, any method of sample preparation may be used
that produces a label corresponding to a molecule of cardiac
troponin that is wished to be measured, where the label is
detectable in the instruments described herein. As is known in the
art, sample preparation in which a label is added to one or more
particles may be performed in a homogeneous or heterogeneous
format. In some embodiments, the sample preparation is formed in a
homogenous format. In analyzer system employing a homogenous
format, unbound label is not removed from the sample. See, e.g.,
U.S. patent application Ser. No. 11/048,660, incorporated by
reference herein in its entirety. In some embodiments, the particle
or particles of interest are labeled by addition of labeled
antibody or antibodies that bind to the particle or particles of
interest.
[0105] In some embodiments, a heterogeneous assay format is used,
where, typically, a step is employed for removing unbound label.
Such assay formats are well-known in the art. One particularly
useful assay format is a sandwich assay, e.g., a sandwich
immunoassay. In this format, the molecule of interest, e.g., marker
of a biological state, is captured, e.g., on a solid support, using
a capture binding partner. Unwanted molecules and other substances
may then optionally be washed away, followed by binding of a label
comprising a detection binding partner and a detectable label,
e.g., fluorescent moiety. Further washes remove unbound label, then
the detectable label is released, usually though not necessarily
still attached to the detection binding partner. In alternative
embodiments, sample and label are added to the capture binding
partner without a wash in between, e.g., at the same time. Other
variations will be apparent to one of skill in the art.
[0106] In some embodiments, the method for detecting troponin
particles uses a sandwich assay with antibodies, e.g., monoclonal
antibodies as capture binding partners. The method comprises
binding troponin molecules in a sample to a capture antibody that
is immobilized on a binding surface, and binding the detection
antibody to the troponin molecule to form a "sandwich" complex. The
detection antibody comprises a detectable fluorescent label, as
described herein, which is detected, e.g., using the single
molecule analyzers of the invention. Both the capture and detection
antibodies specifically bind troponin. Many example of sandwich
immunoassays are known, and some are described in U.S. Pat. No.
4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et
al., both of which are incorporated herein by reference. Further
examples specific to cardiac troponin are described in the
Examples.
[0107] The capture binding partner may be attached to a solid
support, e.g., a microtiter plate or paramagnetic beads. In some
embodiments, the invention provides a binding partner for a cardiac
troponin attached to a paramagnetic bead. Any suitable binding
partner that is specific for the type of cardiac troponin that it
is wished to capture may be used. The binding partner may be an
antibody, e.g., a monoclonal antibody. The antibody may be specific
for free cardiac troponin (cTnI or cTnT) or for complexed cardiac
troponin, modified cardiac troponin, or fragments of cardiac
troponin, as described herein, or specific to all or substantially
all forms of cardiac troponin, e.g., cTnI or cTnT, likely to be
found in the sample of interest. Production and sources of
antibodies to cardiac troponin are described elsewhere herein.
Preferred antibodies for measuring total troponin are those that
not substantially affected by heparin, phosphorylation, oxidation
and troponin complex formation, and that do not cross-react with
skeletal muscle troponin, e.g., troponin I. In some embodiments,
the antibody is specific for a specific region of a cardiac
troponin. In some embodiments, the region includes amino acids
41-49 of human cardiac troponin I. In some embodiments, the region
includes amino acids 87-91 of human cardiac troponin I. Such
antibodies are well-known in the art and are available from, e.g.
BiosPacific, Emeryville, Calif. An example of a capture antibody
useful in embodiments of the invention is an antibody, e.g., a
monoclonal antibody, that reacts with free cardiac troponin I
(cTnI) a.a. 41-49 and cTnI forming complexes with other troponin
components. Preferably, this antibody is not affected by heparin,
phosphorylation, oxidation and troponin complex formation, and does
not cross-react with skeletal muscle troponin I. An exemplary
antibody of this type is Monoclonal Antibody Clone Number
A34650228P, available from BiosPacific, Emeryville, Calif. Another
example of a capture antibody useful in embodiments of the
invention is an antibody, e.g., a monoclonal antibody, that reacts
with free cardiac troponin I (cTnI) a.a. 87-91 and cTnI forming
complexes with other troponin components. Preferably, this antibody
is not affected by heparin, phosphorylation, oxidation and troponin
complex formation, and does not cross-react with skeletal muscle
troponin I. An exemplary antibody of this type is Monoclonal
Antibody Clone Number A34440228P, available from BiosPacific,
Everyville, Calif. It will be appreciated that antibodies
identified herein as useful as a capture antibody may also be
useful as detection antibodies, and vice versa.
[0108] The attachment of the binding partner, e.g., antibody, to
the solid support may be covalent or noncovalent. In some
embodiments, the attachment is noncovalent. An example of a
noncovalent attachment well-known in the art is
biotin-avidin/streptavidin interactions. Thus, in some embodiments,
a solid support, e.g., a microtiter plate or a paramagnetic bead,
is attached to the capture binding partner, e.g., antibody, through
noncovalent attachment, e.g., biotin-avidin/streptavidin
interactions. In some embodiments, the attachment is covalent.
Thus, in some embodiments, a solid support, e.g., a microtiter
plate or a paramagnetic bead, is attached to the capture binding
partner, e.g., antibody, through covalent attachment. Covalent
attachment in which the orientation of the capture antibody is such
that capture of the molecule of interest is optimized is especially
useful. For example, in some embodiments a solid support, e.g., a
microtiter plate or a paramagnetic microparticle, may be used in
which the attachment of the binding partner, e.g., antibody, is an
oriented attachment, e.g., a covalent oriented attachment.
[0109] An exemplary protocol for oriented attachment of an antibody
to a solid support is as follows: IgG is dissolved in 0.1M sodium
acetate buffer, pH 5.5 to a final concentration of 1 mg/ml. An
equal volume of ice-cold 20 mM sodium periodate in 0.1 M sodium
acetate, pH 5.5 is added. The IgG is allowed to oxidize for 1/2
hour on ice. Excess periodate reagent is quenched by the addition
of 0.15 volume of 1 M glycerol. Low molecular weight byproducts of
the oxidation reaction are removed by ultrafiltration. The oxidized
IgG fraction is diluted to a suitable concentration (typically 0.5
micrograms IgG per ml) and reacted with hydrazide-activated
multiwell plates for at least two hours at room temperature.
Unbound IgG is removed by washing the multiwell plate with borate
buffered saline or another suitable buffer. The plate may be dried
for storage, if desired. A similar protocol may be followed for
microbeads if the material of the microbead is suitable for such
attachment.
[0110] In some embodiments, the solid support is a microtiter
plate. In some embodiments, the solid support is a paramagnetic
bead. An exemplary paramagnetic bead is Streptavidin C1 (Dynal,
650.01-03). Other suitable beads will be apparent to those of skill
in the art. Methods for attachment of antibodies to paramagnetic
beads are well-known in the art. One example is given in the
Examples.
[0111] The cardiac troponin of interest is contacted with the
capture binding partner, e.g., capture antibody immobilized on a
solid support. Some sample preparation may be used; e.g.,
preparation of serum from blood samples or concentration procedures
before the sample is contacted with the capture antibody. Protocols
for binding of proteins in immunoassays are well-known in the art
and are included in the Examples.
[0112] The time allowed for binding will vary depending on the
conditions; it will be apparent that shorter binding times are
desirable in some settings, especially in a clinical setting. The
use of, e.g., paramagnetic beads can reduce the time required for
binding. In some embodiments, the time allowed for binding of the
protein of interest to the capture binding partner, e.g., antibody,
is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than
about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some
embodiments, the time allowed for binding of the protein of
interest to the capture binding partner, e.g., antibody, is less
than about 60 minutes. In some embodiments, the time allowed for
binding of the protein of interest to the capture binding partner,
e.g., antibody, is less that about 40 minutes. In some embodiments,
the time allowed for binding of the protein of interest to the
capture binding partner, e.g., antibody, is less that about 30
minutes. In some embodiments, the time allowed for binding of the
protein of interest to the capture binding partner, e.g., antibody,
is less that about 20 minutes. In some embodiments, the time
allowed for binding of the protein of interest to the capture
binding partner, e.g., antibody, is less that about 15 minutes. In
some embodiments, the time allowed for binding of the protein of
interest to the capture binding partner, e.g., antibody, is less
that about 10 minutes. In some embodiments, the time allowed for
binding of the protein of interest to the capture binding partner,
e.g., antibody, is less that about 5 minutes.
[0113] In some embodiments, following the binding of the troponin
particles to the capture binding partner, e.g., capture antibody,
particles that may have bound nonspecifically, as well as other
unwanted substances in the sample, are washed away leaving
substantially only specifically bound troponin particles. In other
embodiments, no wash is used between additions of sample and label;
it will be appreciated that this reduces sample preparation time
even further. Thus, in some embodiments, the time allowed for both
binding of the protein of interest to the capture binding partner,
e.g., antibody, and binding of the label to the protein of
interest, is less that about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or
less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In
some embodiments, the time allowed for both binding of the protein
of interest to the capture binding partner, e.g., antibody, and
binding of the label to the protein of interest, is less that about
60 minutes. In some embodiments, the time allowed for both binding
of the protein of interest to the capture binding partner, e.g.,
antibody, and binding of the label to the protein of interest, is
less than about 40 minutes. In some embodiments, the time allowed
for both binding of the protein of interest to the capture binding
partner, e.g., antibody, and binding of the label to the protein of
interest, is less than about 30 minutes. In some embodiments, the
time allowed for both binding of the protein of interest to the
capture binding partner, e.g., antibody, and binding of the label
to the protein of interest, is less than about 20 minutes. In some
embodiments, the time allowed for both binding of the protein of
interest to the capture binding partner, e.g., antibody, and
binding of the label to the protein of interest, is less than about
15 minutes. In some embodiments, the time allowed for both binding
of the protein of interest to the capture binding partner, e.g.,
antibody, and binding of the label to the protein of interest, is
less than about 10 minutes. In some embodiments, the time allowed
for both binding of the protein of interest to the capture binding
partner, e.g., antibody, and binding of the label to the protein of
interest, is less than about 5 minutes.
[0114] Some immunoassay diagnostic reagents including the capture
and signal antibodies used to measure the target analytes may be
derived from the sera of animals. Endogenous human heterophilic
antibodies, or human anti-animal antibodies, which have the ability
to bind to immunoglobulins of other species, are present in the
serum or plasma of more than 10% of patients. These circulating
heterophile antibodies may interfere with immunoassay measurements.
In sandwich immunoassays, these heterophilic antibodies can either
bridge the capture and detection (diagnostic) antibodies, thereby
producing a false-positive signal, or they may block the binding of
the diagnostic antibodies, thereby producing a false-negative
signal. In competitive immunoassays, the heterophile antibodies may
bind to the analytic antibody and inhibit its binding to the
troponin. They also may either block or augment the separation of
the antibody-troponin complex from free troponin, especially when
antispecies antibodies are used in the separation systems.
Therefore, the impact of these heterophile antibody interferences
are difficult to predict. Thus, it would be advantageous to block
the binding of any heterophilic antibodies. In some embodiments of
the invention, the immunoassay includes the step of depleting the
sample of heterophile antibodies using one or more heterophile
antibody blockers. Methods for removing heterophile antibodies from
samples that are to be tested in immunoassays are known and
include: heating the specimen in a sodium acetate buffer, pH 5.0,
for 15 minutes at 90.degree. C. and centrifuging at 1200 g for 10
minutes, or the heterophile antibodies can be precipitated using
polyethylene glycol (PEG); immunoextracting the interfering
heterophile immunoglobulins from the specimen using protein A or
protein G; or adding nonimmune mouse IgG. Embodiments of the
methods of the invention contemplate preparing the sample prior to
analysis with the single molecule detector. The appropriateness of
the method of pretreatment may be determined. Biochemicals to
minimize immunoassay interference caused by heterophile antibodies
are commercially available. For example, a product called MAK33,
which is an IgG1 monoclonal antibody to h-CK-MM, may be obtained
from Boehringer Mannheim. The MAK33 plus product contains a
combination of IgG1 and IgG1-Fab. The polyMAK33 contains IgG1-Fab
polymerized with IgG1, and the polyMAC 2b/2a contains IgG2a-Fab
polymerized with IgG2b. A second commercial source of biochemicals
to neutralize heterophile antibodies is Immunoglobulin Inhibiting
Reagent marketed by Bioreclamation Inc, East Meadow, N.Y. This
product is a preparation of immunoglobulins (IgG and IgM) from
multiple species, mainly murine IgG2a, IgG2b, and IgG3 from Balb/c
mice. In some embodiments the heterophile antibody may be
immunoextracted from the sample using methods known in the art e.g.
depleting the sample of the heterophile antibody by binding the
interfering antibody to protein A or G. In some embodiments, the
heterophile antibody is neutralized using one or more heterophile
antibody blockers. Heterophile blockers may be selected from the
group consisting of anti-isotype heterophile antibody blockers,
anti-idiotype heterophile antibody blockers, and anti-anti-idiotype
heterophile antibody blockers. In some embodiments a combination of
heterophile antibody blockers may be used.
[0115] Label is added either with or following the addition of
sample and washing. Protocols for binding of antibody and other
immunolabels to proteins and other molecules are well-known in the
art. If the label binding step is separate from capture binding,
the time allowed for label binding can be important, e.g., in the
clinical setting. In some embodiments, the time allowed for binding
of the protein of interest to the label, e.g., antibody-dye, is
less than about 12, 10, 8, 6, 4, 3, 2, or 1 hours, or less than
about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some
embodiments, the time allowed for binding of the protein of
interest to the label, e.g., antibody-dye, is less than about 60
minutes. In some embodiments, the time allowed for binding of the
protein of interest to the label, e.g., antibody-dye, is less than
about 40 minutes. In some embodiments, the time allowed for binding
of the protein of interest to the label, e.g., antibody-dye, is
less than about 30 minutes. In some embodiments, the time allowed
for binding of the protein of interest to the label, e.g.,
antibody-dye, is less than about 20 minutes. In some embodiments,
the time allowed for binding of the protein of interest to the
label, e.g., antibody-dye, is less than about 15 minutes. In some
embodiments, the time allowed for binding of the protein of
interest to the label, e.g., antibody-dye, is less than about 10
minutes. In some embodiments, the time allowed for binding of the
protein of interest to the label, e.g., antibody-dye, is less than
about 5 minutes. Excess label is removed by washing.
[0116] Label is then eluted from the protein of interest. Preferred
elution buffers are effective in releasing the label without
generating significant background. It is also useful if the elution
buffer is bacteriostatic. Elution buffers of use in the invention
include a chaotrope, e.g., urea or a guanidinium compound; a
buffer, e.g., borate buffered saline; a protein carrier, e.g., an
albumin, such as human, bovine, or fish albumin, or an IgG, to coat
the wall of the capillary tube in the detection instrument; and a
surfactant, e.g., an ionic or nonionic detergent, selected so as to
produce a relatively low background, e.g., Tween 20, Triton X-100,
or SDS.
[0117] The elution buffer/label aliquot that is sampled into the
single molecule detector is referred to as the "processing sample,"
to distinguish it from the original sample which was obtained from
an individual.
[0118] In another embodiment, the solid phase binding assay may
employ a competitive binding assay format. One such method
comprises a) competitively binding to a capture antibody
immobilized on a binding surface i) a troponin particle in a sample
and ii) a labeled analog of the troponin particle comprising a
detectable label (the detection reagent) and b) measuring the
amount of the label using a single particle analyzer. Another such
method comprises a) competitively binding to an antibody having a
detectable label (the detection reagent) i) a troponin particle in
a sample and ii) an analog of troponin particle that is immobilized
on a binding surface (the capture reagent) and b) measuring the
amount of the label using a single particle analyzer. An "analog of
a troponin" refers, herein, to a species that competes with
troponin for binding to a capture antibody. Examples of competitive
immunoassays are disclosed in U.S. Pat. No. 4,235,601 to Deutsch et
al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535
to Buechler et al., all of which are incorporated herein by
reference.
[0119] C. Detection of Troponin and Determination of
Concentration
[0120] Following elution, the label is run through a single
molecule detector in e.g., the elution buffer. A processing sample
may contain no label, a single label, or a plurality of labels. The
number of labels corresponds or is proportional to (if dilutions or
fractions of samples are used) the number of molecules of cardiac
troponin captured during the capture step.
[0121] Any suitable single molecule detector capable of detecting
the label used with the protein of interest may be used. Suitable
single molecule detectors are described herein. Typically the
detector will be part of a system that includes an automatic
sampler for sampling prepared samples, and, optionally, a recovery
system to recover samples.
[0122] In some embodiments, the processing sample is analyzed in a
single molecule analyzer that utilizes a capillary flow system, and
that includes a capillary flow cell, a laser to illuminate an
interrogation space in the capillary through which processing
sample is passed, a detector to detect radiation emitted from the
interrogation space, and a source of motive force to move a
processing sample through the interrogation space. In some
embodiments, the single molecule analyzer further comprises a
microscope objective lens that collects light emitted from
processing sample as it passes through the interrogation space,
e.g., a high numerical aperture microscope objective. In some
embodiments, the laser and detector are in a confocal arrangement.
In some embodiments, the laser is a continuous wave laser. In some
embodiments, the detector is a avalanche photodiode detector. In
some embodiments, the source of motive force is a pump to provide
pressure. In some embodiments, the invention provides an analyzer
system that includes a sampling system capable of automatically
sampling a plurality of samples providing a fluid communication
between a sample container and the interrogation space. In some
embodiments, the interrogation space has a volume of between about
0.001 and 500 pL, or between about 0.01 pL and 100 pL, or between
about 0.01 pL and 10 pL, or between about 0.01 pL and 5 pL, or
between about 0.01 pL and 0.5 pL, or between about 0.02 pL and
about 300 pL, or between about 0.02 pL and about 50 pL or between
about 0.02 pL and about 5 pL or between about 0.02 pL and about 0.5
pL or between about 0.02 pL and about 2 pL, or between about 0.05
pL and about 50 pL, or between about 0.05 pL and about 5 pL, or
between about 0.05 pL and about 0.5 pL, or between about 0.05 pL
and about 0.2 pL, or between about 0.1 pL and about 25 pL. In some
embodiments, the interrogation space has a volume between about
0.004 pL and 100 pL. In some embodiments, the interrogation space
has a volume between about 0.02 pL and 50 pL. In some embodiments,
the interrogation space has a volume between about 0.001 pL and 10
pL. In some embodiments, the interrogation space has a volume
between about 0.001 pL and 10 pL. In some embodiments, the
interrogation space has a volume between about 0.01 pL and 5 pL. In
some embodiments, the interrogation space has a volume between
about 0.02 pL and about 5 pL. In some embodiments, the
interrogation space has a volume between about 0.05 pL and 5 pL. In
some embodiments, the interrogation space has a volume between
about 0.05 pL and 10 pL. In some embodiments, the interrogation
space has a volume between about 0.5 pL and about 5 pL. In some
embodiments, the interrogation space has a volume between about
0.02 pL and about 0.5 pL.
[0123] In some embodiments, the single molecule detector used in
the methods of the invention utilizes a capillary flow system, and
includes a capillary flow cell, a continuous wave laser to
illuminate an interrogation space in the capillary through which
processing sample is passed, a high numerical aperture microscope
objective lens that collects light emitted from processing sample
as it passes through the interrogation space, an avalanche
photodiode detector to detect radiation emitted from the
interrogation space, and a pump to provide pressure to move a
processing sample through the interrogation space, where the
interrogation space is between about 0.02 pL and about 50 pL. In
some embodiments, the single molecule detector used in the methods
of the invention utilizes a capillary flow system, and includes a
capillary flow cell, a continuous wave laser to illuminate an
interrogation space in the capillary through which processing
sample is passed, a high numerical aperture microscope objective
lens that collects light emitted from processing sample as it
passes through the interrogation space wherein the lens has a
numerical aperture of at least about 0.8, an avalanche photodiode
detector to detect radiation emitted from the interrogation space,
and a pump to provide pressure to move a processing sample through
the interrogation space, where the interrogation space is between
about 0.004 pL and about 100 pL. In some embodiments, the single
molecule detector used in the methods of the invention utilizes a
capillary flow system, and includes a capillary flow cell, a
continuous wave laser to illuminate an interrogation space in the
capillary through which processing sample is passed, a high
numerical aperture microscope objective lens that collects light
emitted from processing sample as it passes through the
interrogation space wherein the lens has a numerical aperture of at
least about 0.8, an avalanche photodiode detector to detect
radiation emitted from the interrogation space, and a pump to
provide pressure to move a processing sample through the
interrogation space, where the interrogation space is between about
0.05 pL and about 10 pL. In some embodiments, the single molecule
detector used in the methods of the invention utilizes a capillary
flow system, and includes a capillary flow cell, a continuous wave
laser to illuminate an interrogation space in the capillary through
which processing sample is passed, a high numerical aperture
microscope objective lens that collects light emitted from
processing sample as it passes through the interrogation space
wherein the lens has a numerical aperture of at least about 0.8, an
avalanche photodiode detector to detect radiation emitted from the
interrogation space, and a pump to provide pressure to move a
processing sample through the interrogation space, where the
interrogation space is between about 0.05 pL and about 5 pL. In
some embodiments, the single molecule detector used in the methods
of the invention utilizes a capillary flow system, and includes a
capillary flow cell, a continuous wave laser to illuminate an
interrogation space in the capillary through which processing
sample is passed, a high numerical aperture microscope objective
lens that collects light emitted from processing sample as it
passes through the interrogation space wherein the lens has a
numerical aperture of at least about 0.8, an avalanche photodiode
detector to detect radiation emitted from the interrogation space,
and a pump to provide pressure to move a processing sample through
the interrogation space, where the interrogation space is between
about 0.5 pL and about 5 pL.
[0124] In some embodiments, the single molecule detector is capable
of determining a concentration for a molecule of interest in a
sample where sample may range in concentration over a range of at
least about 100-fold, or 1000-fold, or 10,000-fold, or
100,000-fold, or 300,00-fold, or 1,000,000-fold, or
10,000,000-fold, or 30,000,000-fold.
[0125] In some embodiments, the methods of the invention utilize a
single molecule detector capable detecting a difference of less
than about 50%, 40%, 30%, 20%, 15%, or 10% in concentration of an
analyte between a first sample and a second sample that are
introduced into the detector, where the volume of the first sample
and said second sample introduced into the analyzer is less than
about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1
ul, and wherein the analyte is present at a concentration of less
than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2,
or 1 femtomolar. In some embodiments, the methods of the invention
utilize a single molecule detector capable detecting a difference
of less than about 50% in concentration of an analyte between a
first sample and a second sample that are introduced into the
detector, where the volume of the first sample and said second
sample introduced into the analyzer is less than about 100 ul, and
wherein the analyte is present at a concentration of less than
about 100 femtomolar. In some embodiments, the methods of the
invention utilize a single molecule detector capable detecting a
difference of less than about 40% in concentration of an analyte
between a first sample and a second sample that are introduced into
the detector, where the volume of the first sample and said second
sample introduced into the analyzer is less than about 50 ul, and
wherein the analyte is present at a concentration of less than
about 50 femtomolar. In some embodiments, the methods of the
invention utilize a single molecule detector capable detecting a
difference of less than about 20% in concentration of an analyte
between a first sample and a second sample that are introduced into
the detector, where the volume of the first sample and said second
sample introduced into the analyzer is less than about 20 ul, and
wherein the analyte is present at a concentration of less than
about 20 femtomolar. In some embodiments, the methods of the
invention utilize a single molecule detector capable detecting a
difference of less than about 20% in concentration of an analyte
between a first sample and a second sample that are introduced into
the detector, where the volume of the first sample and said second
sample introduced into the analyzer is less than about 10 ul, and
wherein the analyte is present at a concentration of less than
about 10 femtomolar. In some embodiments, the methods of the
invention utilize a single molecule detector capable detecting a
difference of less than about 20% in concentration of an analyte
between a first sample and a second sample that are introduced into
the detector, where the volume of the first sample and said second
sample introduced into the analyzer is less than about 5 ul, and
wherein the analyte is present at a concentration of less than
about 5 femtomolar.
[0126] The single molecule detector and systems are described in
more detail below. Further embodiments of single molecule analyzers
useful in the methods of the invention, such as detectors with more
than one interrogation window, detectors utilize electrokinetic or
electrophoretic flow, and the like, may be found in U.S. patent
application Ser. No. 11/048,660, incorporated by reference herein
in its entirety.
[0127] Between runs the instrument may be washed. A wash buffer
that maintains the salt and surfactant concentrations of the sample
may be used in some embodiments to maintain the conditioning of the
capillary; i.e., to keep the capillary surface relatively constant
between samples to reduce variability.
[0128] A feature that contributes to the extremely high sensitivity
of the instruments and methods of the invention is the method of
detecting and counting labels, which, in some embodiments, are
attached to single molecules to be detected or, more typically,
correspond to a single molecule to be detected. Briefly, the
processing sample flowing through the capillary is effectively
divided into a series of detection events, by subjecting a given
interrogation space of the capillary to EM radiation from a laser
that emits light at an appropriate excitation wavelength for the
fluorescent moiety used in the label for a predetermined period of
time, and detecting photons emitted during that time. Each
predetermined period of time is a "bin." If the total number of
photons detected in a given bin exceeds a predetermined threshold
level, a detection event is registered for that bin, i.e., a label
has been detected. If the total number of photons is not at the
predetermined threshold level, no detection event is registered. In
some embodiments, processing sample concentration is dilute enough
that, for a large percentage of detection events, the detection
event represents only one label passing through the window, which
corresponds to a single molecule of interest in the original
sample, that is, few detection events represent more than one label
in a single bin. In some embodiments, further refinements are
applied to allow greater concentrations of label in the processing
sample to be detected accurately, i.e., concentrations at which the
probability of two or more labels being detected as a single
detection event is no longer insignificant.
[0129] Although other bin times may be used without departing from
the scope of the present invention, in some embodiments the bin
times are selected in the range of about 1 microsecond to about 5
ms. In some embodiments, the bin time is more than about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,
250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000,
4000, or 5000 microseconds. In some embodiments, the bin time is
less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,
70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900,
1000, 2000, 3000, 4000, or 5000 microseconds. In some embodiments,
the bin time is about 1 to 1000 microseconds. In some embodiments,
the bin time is about 1 to 750 microseconds. In some embodiments,
the bin time is about 1 to 500 microseconds. In some embodiments,
the bin time is about 1 to 250 microseconds. In some embodiments,
the bin time is about 1 to 100 microseconds. In some embodiments,
the bin time is about 1 to 50 microseconds. In some embodiments,
the bin time is about 1 to 40 microseconds. In some embodiments,
the bin time is about 1 to 30 microseconds. In some embodiments,
the bin time is about 1 to 500 microseconds. In some embodiments,
the bin time is about 1 to 20 microseconds. In some embodiments,
the bin time is about 1 to 10 microseconds. In some embodiments,
the bin time is about 1 to 500 microseconds. In some embodiments,
the bin time is about 1 to 5 microseconds. In some embodiments, the
bin time is about 5 to 500 microseconds. In some embodiments, the
bin time is about 5 to 250 microseconds. In some embodiments, the
bin time is about 5 to 100 microseconds. In some embodiments, the
bin time is about 5 to 50 microseconds. In some embodiments, the
bin time is about 5 to 20 microseconds. In some embodiments, the
bin time is about 5 to 10 microseconds. In some embodiments, the
bin time is about 10 to 500 microseconds. In some embodiments, the
bin time is about 10 to 250 microseconds. In some embodiments, the
bin time is about 10 to 100 microseconds. In some embodiments, the
bin time is about 10 to 50 microseconds. In some embodiments, the
bin time is about 10 to 30 microseconds. In some embodiments, the
bin time is about 10 to 20 microseconds. In some embodiments, the
bin time is about 5 microseconds. In some embodiments, the bin time
is about 5 microseconds. In some embodiments, the bin time is about
6 microseconds. In some embodiments, the bin time is about 7
microseconds. In some embodiments, the bin time is about 8
microseconds. In some embodiments, the bin time is about 9
microseconds. In some embodiments, the bin time is about 10
microseconds. In some embodiments, the bin time is about 11
microseconds. In some embodiments, the bin time is about 12
microseconds. In some embodiments, the bin time is about 13
microseconds. In some embodiments, the bin time is about 14
microseconds. In some embodiments, the bin time is about 5
microseconds. In some embodiments, the bin time is about 15
microseconds. In some embodiments, the bin time is about 16
microseconds. In some embodiments, the bin time is about 17
microseconds. In some embodiments, the bin time is about 18
microseconds. In some embodiments, the bin time is about 19
microseconds. In some embodiments, the bin time is about 20
microseconds. In some embodiments, the bin time is about 25
microseconds. In some embodiments, the bin time is about 30
microseconds. In some embodiments, the bin time is about 40
microseconds. In some embodiments, the bin time is about 50
microseconds. In some embodiments, the bin time is about 100
microseconds. In some embodiments, the bin time is about 250
microseconds. In some embodiments, the bin time is about 500
microseconds. In some embodiments, the bin time is about 750
microseconds. In some embodiments, the bin time is about 1000
microseconds.
[0130] In some embodiments, the background noise level is
determined from the mean noise level, or the root-mean-square
noise. In other cases, a typical noise value or a statistical value
is chosen. In most cases, the noise is expected to follow a Poisson
distribution. Thus, in some embodiments, determining the
concentration of a particle-label complex in a sample comprises
determining the background noise level.
[0131] Thus, as a label flows through the capillary flow cell, it
is irradiated by the laser beam to generate a burst of photons. The
photons emitted by the label are discriminated from background
light or background noise emission by considering only the bursts
of photons that have energy above a predetermined threshold energy
level which accounts for the amount of background noise that is
present in the sample. Background noise typically comprises low
frequency emission produced, for example, by the intrinsic
fluorescence of non-labeled particles that are present in the
sample, the buffer or diluent used in preparing the sample for
analysis, Raman scattering and electronic noise. In some
embodiments, the value assigned to the background noise is
calculated as the average background signal noise detected in a
plurality of bins, which are measurements of photon signals that
are detected in an interrogation space during a predetermined
length of time. Thus in some embodiments, background noise is
calculated for each sample as a number specific to that sample.
[0132] Given the value for the background noise, the threshold
energy level can be assigned. As discussed above, the threshold
value is determined to discriminate true signals (due to
fluorescence of a label) from the background noise. Care must be
taken in choosing a threshold value such that the number of false
positive signals from random noise is minimized while the number of
true signals which are rejected is also minimized. Methods for
choosing a threshold value include determining a fixed value above
the noise level and calculating a threshold value based on the
distribution of the noise signal. In one embodiment, the threshold
is set at a fixed number of standard deviations above the
background level. Assuming a Poisson distribution of the noise,
using this method one can estimate the number of false positive
signals over the time course of the experiment. In some
embodiments, the threshold level is calculated as a value of 4
sigma above the background noise. For example, given an average
background noise level of 200 photons, the analyzer system
establishes a threshold level of 4 200 above the average
background/noise level of 200 photons to be 256 photons. Thus, in
some embodiments, determining the concentration of a label in a
sample includes establishing the threshold level above which photon
signals represent the presence of a label. Conversely, photon
signals that have an energy level that is not greater than that of
the threshold level indicate the absence of a label.
[0133] Many bin measurements are taken to determine the
concentration of a sample, and the absence or presence of a label
is ascertained for each bin measurement. Typically, 60,000
measurements or more can made in one minute (e.g., in embodiments
in which the bin size is 1 ms--for smaller bin sizes the number of
measurements is correspondingly larger, e.g., 6,000,000
measurements per minute for a bin size of 10 microseconds). Thus,
no single measurement is crucial and the method provides for a high
margin of error. The bins that are determined not to contain a
label ("no" bins) are discounted and only the measurements made in
the bins that are determined to contain label ("yes" bins) are
accounted in determining the concentration of the label in the
processing sample. Discounting measurements made in the "no" bins
or bins that are devoid of label increases the signal to noise
ratio and the accuracy of the measurements. Thus, in some
embodiments, determining the concentration of a label in a sample
comprises detecting the bin measurements that reflect the presence
of a label.
[0134] The signal to noise ratio or the sensitivity of the analyzer
system can be increased by minimizing the time that background
noise is detected during a bin measurement in which a
particle-label complex is detected. For example, in a bin
measurement lasting 1 millisecond during which one particle-label
complex is detected when passing across an interrogation space
within 250 microseconds, 750 microseconds of the 1 millisecond are
spent detecting background noise emission. The signal to noise
ratio can be improved by decreasing the bin time. In some
embodiments, the bin time is 1 millisecond. In other embodiments,
the bin time is 750, 500, 250 microseconds, 100 microseconds, 50
microseconds, 25 microseconds or 10 microseconds. Other bin times
are as described herein.
[0135] Other factors that affect measurements are the brightness or
dimness of the fluorescent moiety, the flow rate, and the power of
the laser. Various combinations of the relevant factors that allow
for detection of label will be apparent to those of skill in the
art. In some embodiments, the bin time is adjusted without changing
the flow rate. It will be appreciated by those of skill in the art
that as bin time decreases, laser power output directed at the
interrogation space must increase to maintain a constant total
energy applied to the interrogation space during the bin time. For
example, if bin time is decreased from 1000 microseconds to 250
microseconds, as a first approximation, laser power output must be
increased approximately four-fold. These settings allow for the
detection of the same number of photons in a 250 .mu.s as the
number of photons counted during the 1000 .mu.s given the previous
settings, and allow for faster analysis of sample with lower
backgrounds and thus greater sensitivity. In addition, flow rates
may be adjusted in order to speed processing of sample. These
numbers are merely exemplary, and the skilled practitioner can
adjust the parameters as necessary to achieve the desired
result.
[0136] In some embodiments, the interrogation space encompasses the
entire cross-section of the sample stream. When the interrogation
space encompasses the entire cross-section of the sample stream,
only the number of labels counted and the volume passing through a
cross-section of the sample stream in a set length of time are
needed to calculate the concentration of the label in the
processing sample. In some embodiments, the interrogation space can
be defined to be smaller than the cross-sectional area of sample
stream by, for example, the interrogation space is defined by the
size of the spot illuminated by the laser beam. In some
embodiments, the interrogation space can be defined by adjusting
the apertures 306 (FIG. 1A) or 358 and 359 (FIG. 1B) of the
analyzer and reducing the illuminated volume that is imaged by the
objective lens to the detector. In the embodiments when the
interrogation space is defined to be smaller than the
cross-sectional area of sample stream, the concentration of the
label can be determined by interpolation of the signal emitted by
the complex from a standard curve that is generated using one or
more samples of known standard concentrations. In yet other
embodiments, the concentration of the label can be determined by
comparing the measured particles to an internal label standard. In
embodiments when a diluted sample is analyzed, the dilution factor
is accounted in calculating the concentration of the molecule of
interest in the starting sample.
[0137] As discussed above, when the interrogation space encompasses
the entire cross-section of the sample stream, only the number of
labels counted passing through a cross-section of the sample stream
in a set length of time (bin) and the volume of sample that was
interrogated in the bin are needed to calculate the concentration
the sample. The total number of labels contained in the "yes" bins
is determined and related to the sample volume represented by the
total number of bins used in the analysis to determine the
concentration of labels in the processing sample. Thus, in one
embodiment, determining the concentration of a label in a
processing sample comprises determining the total number of labels
detected "yes" bins and relating the total number of detected
labels to the total sample volume that was analyzed. The total
sample volume that is analyzed is the sample volume that is passed
through the capillary flow cell and across the interrogation space
in a specified time interval. Alternatively, the concentration of
the label complex in a sample is determined by interpolation of the
signal emitted by the label in a number of bins from a standard
curve that is generated by determining the signal emitted by labels
in the same number of bins by standard samples containing known
concentrations of the label.
[0138] In some embodiments, the number of individual labels that
are detected in a bin is related to the relative concentration of
the particle in the processing sample. At relatively low
concentrations, for example at concentrations below about 10 -16 M
the number of labels is proportional to the photon signal that is
detected in a bin. Thus, at low concentrations of label the photon
signal is provided as a digital signal. At relatively higher
concentrations, for example at concentrations greater than about 10
-16 M, the proportionality of photon signal to a label is lost as
the likelihood of two or more labels crossing the interrogation
space at about the same time and being counted as one becomes
significant. Thus, in some embodiments, individual particles in a
sample of a concentration greater than about 10.sup.-16 M are
resolved by decreasing the length of time of the bin
measurement.
[0139] Alternatively, in other embodiments, the total the photon
signal that is emitted by a plurality of particles that are present
in any one bin is detected. These embodiments allow for single
molecule detectors of the invention wherein the dynamic range is at
least 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, or more than 8
logs.
[0140] "Dynamic range," as that term is used herein, refers to the
range of sample concentrations that may be quantitated by the
instrument without need for dilution or other treatment to alter
the concentration of successive samples of differing
concentrations, where concentrations are determined with an
accuracy appropriate for the intended use. For example, if a
microtiter plate contains a sample of 1 femtomolar concentration
for an analyte of interest in one well, a sample of 10,000
femtomolar concentration for an analyte of interest in another
well, and a sample of 100 femtomolar concentration for the analyte
in a third well, an instrument with a dynamic range of at least 4
logs and a lower limit of quantitation of 1 femtomolar is able to
accurately quantitate the concentration of all the samples without
the need for further treatment to adjust concentration, e.g.,
dilution. Accuracy may be determined by standard methods, e.g.,
using a series of standards of concentrations that span the dynamic
range and constructing a standard curve. Standard measures of fit
of the resulting standard curve may be used as a measure of
accuracy, e.g., an r.sup.2 greater than about 0.7, 0.75, 0.8, 0.85,
0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.
[0141] Increased dynamic range is achieved by altering the manner
in which data from the detector is analyzed, and/or by the use of
an attenuator between the detector and the interrogation space. At
the low end of the range, where processing sample is sufficiently
dilute that each detection event, i.e., each burst of photons above
a threshold level in a bin (the "event photons"), likely represents
only one label, the data is analyzed to count detection events as
single molecules. I.e., each bin is analyzed as a simple "yes" or
"no" for the presence of label, as described above. For a more
concentrated processing sample, where the likelihood of two or more
labels occupying a single bin becomes significant, the number of
event photons in a significant number of bins is found to be
substantially greater than the number expected for a single label,
e.g., the number of event photons in a significant number of bins
corresponds to two-fold, three-fold, or more, than the number of
event photons expected for a single label. For these samples, the
instrument changes its method of data analysis to one of
integrating the total number of event photons for the bins of the
processing sample. This total will be proportional to the total
number of labels that were in all the bins. For an even more
concentrated processing sample, where many labels are present in
most bins, background noise becomes an insignificant portion of the
total signal from each bin, and the instrument changes its method
of data analysis to one of counting total photons per bin
(including background). An even further increase in dynamic range
can be achieved by the use of an attenuator between the flow cell
and the detector, when concentrations are such that the intensity
of light reaching the detector would otherwise exceed the capacity
of the detector for accurately counting photons, i.e., saturate the
detector.
[0142] The instrument may include a data analysis system that
receives input from the detector and determines the appropriate
analysis method for the sample being run, and outputs values based
on such analysis. The data analysis system may further output
instructions to use or not use an attenuator, if an attenuator is
included in the instrument.
[0143] By utilizing such methods, the dynamic range of the
instrument can be dramatically increased. Thus, in some
embodiments, the instrument is capable of measuring concentrations
of samples over a dynamic range of more than about 1000 (3 log),
10,000 (4 log), 100,000 (5 log), 350,000 (5.5 log), 1,000,000 (6
log), 3,500,000 (6.5 log), 10,000,000 (7 log), 35,000,000 (7.5
log), or 100,000,000 (8 log). In some embodiments, the instrument
is capable of measuring concentrations of samples over a dynamic
range of more than about 100,000 (5 log). In some embodiments, the
instrument is capable of measuring concentrations of samples over a
dynamic range of more than about 1,000,000 (6 log). In some
embodiments, the instrument is capable of measuring concentrations
of samples over a dynamic range of more than about 10,000,000 (7
log). In some embodiments, the instrument is capable of measuring
the concentrations of samples over a dynamic range of from about
1-10 femtomolar to at least about 1000; 10,000; 100,000; 350,000;
1,000,000; 3,500,000; 10,000,000, or 35,000,000 femtomolar. In some
embodiments, the instrument is capable of measuring the
concentrations of samples over a dynamic range of from about 1-10
femtomolar to at least about 10,000 femtomolar. In some
embodiments, the instrument is capable of measuring the
concentrations of samples over a dynamic range of from about 1-10
femtomolar to at least about 100,000 femtomolar. In some
embodiments, the instrument is capable of measuring the
concentrations of samples over a dynamic range of from about 1-10
femtomolar to at least about 1,000,000 femtomolar. In some
embodiments, the instrument is capable of measuring the
concentrations of samples over a dynamic range of from about 1-10
femtomolar to at least about 10,000,000.
[0144] In some embodiments, an analyzer or analyzer system of the
invention is capable of detecting an analyte, e.g., a biomarker at
a limit of detection of less than 1 nanomolar, or 1 picomolar, or 1
femtomolar, or 1 attomolar, or 1 zeptomolar. In some embodiments,
the analyzer or analyzer system is capable of detecting a change in
concentration of the analyte, or of multiple analytes, e.g., a
biomarker or biomarkers, from one sample to another sample of less
than about 0.1, 1, 2, 5, 10, 20, 30, 40, 50, 60, or 80% when the
biomarker is present at a concentration of less than 1 nanomolar,
or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar,
in the samples, and when the size of each of the sample is less
than about 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, or
0.0001 ul. In some embodiments, the analyzer or analyzer system is
capable of detecting a change in concentration of the analyte from
a first sample to a second sample of less than about 20%, when the
analyte is present at a concentration of less than about 1
picomolar, and when the size of each of the samples is less than
about 50 .mu.l. In some embodiments, the analyzer or analyzer
system is capable of detecting a change in concentration of the
analyte from a first sample to a second sample of less than about
20%, when the analyte is present at a concentration of less than
about 100 femtomolar, and when the size of each of the samples is
less than about 50 .mu.l. In some embodiments, the analyzer or
analyzer system is capable of detecting a change in concentration
of the analyte from a first sample to a second sample of less than
about 20%, when the analyte is present at a concentration of less
than about 50 femtomolar, and when the size of each of the samples
is less than about 50 .mu.l. In some embodiments, the analyzer or
analyzer system is capable of detecting a change in concentration
of the analyte from a first sample to a second sample of less than
about 20%, when the analyte is present at a concentration of less
than about 5 femtomolar, and when the size of each of the samples
is less than about 50 .mu.l. In some embodiments, the analyzer or
analyzer system is capable of detecting a change in concentration
of the analyte from a first sample to a second sample of less than
about 20%, when the analyte is present at a concentration of less
than about 5 femtomolar, and when the size of each of the samples
is less than about 5 .mu.l. In some embodiments, the analyzer or
analyzer system is capable of detecting a change in concentration
of the analyte from a first sample to a second sample of less than
about 20%, when the analyte is present at a concentration of less
than about 1 femtomolar, and when the size of each of the samples
is less than about 5 .mu.l.
V. Instruments and Systems Suitable for Highly Sensitive Analysis
of Troponin
[0145] The methods of the invention utilize analytical instruments
of high sensitivity, e.g., single molecule detectors. Such single
molecule detectors include embodiments as hereinafter
described.
[0146] A. Apparatus/System
[0147] In one aspect, the methods described herein utilize an
analyzer system capable of detecting a single particle in a sample.
In one embodiment, the analyzer system is capable of single
particle detection of a fluorescently labeled particle wherein the
analyzer system detects energy emitted by an excited fluorescent
label in response to exposure by an electromagnetic radiation
source when the single particle is present in an interrogation
space defined within a capillary flow cell fluidly connected to the
sampling system of the analyzer system. In a further embodiment of
the analyzer system, the single particle moves through the
interrogation space of the capillary flow cell by means of a motive
force. In another embodiment of the analyzer system, an automatic
sampling system may be included in the analyzer system for
introducing the sample into the analyzer system. In another
embodiment of the analyzer system, a sample preparation system may
be included in the analyzer system for preparing a sample. In a
further embodiment, the analyzer system may contain a sample
recovery system for recovering at least a portion of the sample
after analysis is complete.
[0148] In one aspect, the analyzer system consists of an
electromagnetic radiation source for exciting a single particle
labeled with a fluorescent label. In one embodiment, the
electromagnetic radiation source of the analyzer system is a laser.
In a further embodiment, the electromagnetic radiation source is a
continuous wave laser.
[0149] In a typical embodiment, the electromagnetic radiation
source excites a fluorescent moiety attached to a label as the
label passes through the interrogation space of the capillary flow
cell. In some embodiments, the fluorescent label moiety includes
one or more fluorescent dye molecules. In some embodiments, the
fluorescent label moiety is a quantum dot. Any fluorescent moiety
as described herein may be used in the label.
[0150] A label is exposed to electromagnetic radiation when the
label passes through an interrogation space located within the
capillary flow cell. The interrogation space is typically fluidly
connected to a sampling system. In some embodiments the label
passes through the interrogation space of the capillary flow cell
due to a motive force to advance the label through the analyzer
system. The interrogation space is positioned such that it receives
electromagnetic radiation emitted from the radiation source. In
some embodiments, the sampling system is an automated sampling
system capable of sampling a plurality of samples without
intervention from a human operator.
[0151] The label passes through the interrogation space and emits a
detectable amount of energy when excited by the electromagnetic
radiation source. In one embodiment, an electromagnetic radiation
detector is operably connected to the interrogation space. The
electromagnetic radiation detector is capable of detecting the
energy emitted by the label, e.g., by the fluorescent moiety of the
label.
[0152] In a further embodiment of the analyzer system, the system
further includes a sample preparation mechanism where a sample may
be partially or completely prepared for analysis by the analyzer
system. In some embodiments of the analyzer system, the sample is
discarded after it is analyzed by the system. In other embodiments,
the analyzer system further includes a sample recovery mechanism
whereby at least a portion, or alternatively all or substantially
all, of the sample may be recovered after analysis. In such an
embodiment, the sample can be returned to the origin of the sample.
In some embodiments, the sample can be returned to microtiter wells
on a sample microtiter plate. The analyzer system typically further
consists of a data acquisition system for collecting and reporting
the detected signal.
[0153] B. Single Particle Analyzer
[0154] As shown in FIG. 1A, described herein is one embodiment of
an analyzer system 300. The analyzer system 300 includes an
electromagnetic radiation source 301, a mirror 302, a lens 303, a
capillary flow cell 313, a microscopic objective lens 305, an
aperture 306, a detector lens 307, a detector filter 308, a single
photon detector 309, and a processor 310 operatively connected to
the detector.
[0155] In operation the electromagnetic radiation source 301 is
aligned so that its output 311 is reflected off of a front surface
312 of mirror 302. The lens 303 focuses the beam 311 onto a single
interrogation space (an illustrative example of an interrogation
space 314 is shown in FIG. 2A) in the capillary flow cell 313. The
microscope objective lens 305 collects light from sample particles
and forms images of the beam onto the aperture 306. The aperture
306 affects the fraction of light emitted by the specimen in the
interrogation space of the capillary flow cell 313 that can be
collected. The detector lens 307 collects the light passing through
the aperture 306 and focuses the light onto an active area of the
detector 309 after it passes through the detector filters 308. The
detector filters 308 minimize aberrant noise signals due to light
scatter or ambient light while maximizing the signal emitted by the
excited fluorescent moiety bound to the particle. The processor 310
processes the light signal from the particle according to the
methods described herein.
[0156] In one embodiment, the microscope objective lens 305 is a
high numerical aperture microscope objective. As used herein, "high
numerical aperture lens" include a lens with a numerical aperture
of equal to or greater than 0.6. The numerical aperture is a
measure of the number of highly diffracted image-forming light rays
captured by the objective. A higher numerical aperture allows
increasingly oblique rays to enter the objective lens and thereby
produce a more highly resolved image. Additionally, the brightness
of an image increases with a higher numerical aperture. High
numerical aperture lenses are commercially available from a variety
of vendors, and any one lens having a numerical aperture of equal
to or greater than approximately 0.6 may be used in the analyzer
system. In some embodiments, the lens has a numerical aperture of
about 0.6 to about 1.3. In some embodiments, the lens has a
numerical aperture of about 0.6 to about 1.0. In some embodiments,
the lens has a numerical aperture of about 0.7 to about 1.2. In
some embodiments, the lens has a numerical aperture of about 0.7 to
about 1.0. In some embodiments, the lens has a numerical aperture
of about 0.7 to about 0.9. In some embodiments, the lens has a
numerical aperture of about 0.8 to about 1.3. In some embodiments,
the lens has a numerical aperture of about 0.8 to about 1.2. In
some embodiments, the lens has a numerical aperture of about 0.8 to
about 1.0. In some embodiments, the lens has a numerical aperture
of at least about 0.6. In some embodiments, the lens has a
numerical aperture of at least about 0.7. In some embodiments, the
lens has a numerical aperture of at least about 0.8. In some
embodiments, the lens has a numerical aperture of at least about
0.9. In some embodiments, the lens has a numerical aperture of at
least about 1.0. In some embodiments, the aperture of the
microscope objective lens 305 is approximately 1.25. In an
embodiment where a microscope objective lens 305 of 0.8 is used, a
Nikon 60X/0.8 NA Achromat lens (Nikon, Inc., USA) can be used.
[0157] In some embodiments, the electromagnetic radiation source
301 is a laser that emits light in the visible spectrum. In all
embodiments, the electromagnetic radiation source is set such that
wavelength of the laser is set such that it is of a sufficient
wavelength to excite the fluorescent label attached to the
particle. In some embodiments, the laser is a continuous wave laser
with a wavelength of 639 nm. In other embodiments, the laser is a
continuous wave laser with a wavelength of 532 nm. In other
embodiments, the laser is a continuous wave laser with a wavelength
of 422 nm. In other embodiments, the laser is a continuous wave
laser with a wavelength of 405 nm. Any continuous wave laser with a
wavelength suitable for exciting a fluorescent moiety as used in
the methods and compositions of the invention may be used without
departing from the scope of the invention.
[0158] In a single particle analyzer system 300, as each particle
passes through the beam 311 of the electromagnetic radiation
source, the particle enters into an excited state. When the
particle relaxes from its excited state, a detectable burst of
light is emitted. The excitation-emission cycle is repeated many
times by each particle in the length of time it takes for it to
pass through the beam allowing the analyzer system 300 to detect
tens to thousands of photons for each particle as it passes through
an interrogation space 314. Photons emitted by fluorescent
particles are registered by the detector 309 (FIG. 1A) with a time
delay indicative of the time for the particle label complex to pass
through the interrogation space. The photon intensity is recorded
by the detector 309 and sampling time is divided into bins, which
are uniform, arbitrary, time segments with freely selectable time
channel widths. The number of signals contained in each bin
evaluated. One or a combination of several statistical analytical
methods are employed in order to determine when a particle is
present. Such methods include determining the baseline noise of the
analyzer system and setting a signal strength for the fluorescent
label at a statistical level above baseline noise to eliminate
false positive signals from the detector.
[0159] The electromagnetic radiation source 301 is focused onto a
capillary flow cell 313 of the analyzer system 300 where the
capillary flow cell 313 is fluidly connected to the sample system.
An interrogation space 314 is shown in FIG. 2A. The beam 311 from
the continuous wave electromagnetic radiation source 301 of FIG. 1A
is optically focused to a specified depth within the capillary flow
cell 313. The beam 311 is directed toward the sample-filled
capillary flow cell 313 at an angle perpendicular to the capillary
flow cell 313. The beam 311 is operated at a predetermined
wavelength that is selected to excite a particular fluorescent
label used to label the particle of interest. The size or volume of
the interrogation space 314 is determined by the diameter of the
beam 311 together with the depth at which the beam 311 is focused.
Alternatively, the interrogation space can be determined by running
a calibration sample of known concentration through the analyzer
system.
[0160] When single molecules are detected in the sample
concentration, the beam size and the depth of focus required for
single molecule detection are set and thereby define the size of
the interrogation space 314. The interrogation space 314 is set
such that, with an appropriate sample concentration, only one
particle is present in the interrogation space 314 during each time
interval over which time observations are made. It will be
appreciated that the detection interrogation volume as defined by
the beam is not perfectly spherically shaped, and typically is a
"bow-tie" shape. However, for the purposes of definition, "volumes"
of interrogation spaces are defined herein as the volume
encompassed by a sphere of a diameter equal to the focused spot
diameter of the beam. The focused spot of the beam 311 may have
various diameters without departing from the scope of the present
invention. In some embodiments, the diameter of the focused spot of
the beam is about 1 to about 5, 10, 15, or 20 microns, or about 5
to about 10, 15, or 20 microns, or about 10 to about 20 microns, or
about 10 to about 15 microns. In some embodiments, the diameter of
the focused spot of the beam is about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns. In some
embodiments, the diameter of the focused spot of the beam is about
5 microns. In some embodiments, the diameter of the focused spot of
the beam is about 10 microns. In some embodiments, the diameter of
the focused spot of the beam is about 12 microns. In some
embodiments, the diameter of the focused spot of the beam is about
13 microns. In some embodiments, the diameter of the focused spot
of the beam is about 14 microns. In some embodiments, the diameter
of the focused spot of the beam is about 15 microns. In some
embodiments, the diameter of the focused spot of the beam is about
16 microns. In some embodiments, the diameter of the focused spot
of the beam is about 17 microns. In some embodiments, the diameter
of the focused spot of the beam is about 18 microns. In some
embodiments, the diameter of the focused spot of the beam is about
19 microns. In some embodiments, the diameter of the focused spot
of the beam is about 20 microns.
[0161] In an alternate embodiment of the single particle analyzer
system, more than one electromagnetic radiation source can be used
to excite particles labeled with fluorescent labels of different
wavelengths. In another alternate embodiment, more than one
interrogation space in the capillary flow cell can be used. In
another alternate embodiment, multiple detectors can be employed to
detect different emission wavelengths from the fluorescent labels.
An illustration incorporating each of these alternative embodiments
of an analyzer system is shown in FIG. 1B. These embodiments are
incorporated by reference from previous U.S. patent application
Ser. No. 11/048,660.
[0162] In some embodiments of the analyzer system 300, a motive
force is required to move a particle through the capillary flow
cell 313 of the analyzer system 300. In one embodiment, the motive
force can be a form of pressure. The pressure used to move a
particle through the capillary flow cell can be generated by a
pump. In some embodiments, a Scivex, Inc. HPLC pump can be used. In
some embodiments where a pump is used as a motive force, the sample
can pass through the capillary flow cell at a rate of 1 .mu.L/min
to about 20 .mu.L/min, or about 5 .mu.L/min to about 20 .mu.L/min.
In some embodiments, the sample can pass through the capillary flow
cell at a rate of about 5 .mu.L/min. In some embodiments, the
sample can pass through the capillary flow cell at a rate of about
10 .mu.L/min. In some embodiments, the sample can pass through the
capillary flow cell at a rate of about 15 .mu.L/min. In some
embodiments, the sample can pass through the capillary flow cell at
a rate of about 20 .mu.L/min. In some embodiments, an
electrokinetic force can be used to move the particle through the
analyzer system. Such a method has been previously disclosed and is
incorporated by reference from previous U.S. patent application
Ser. No. 11/048,660.
[0163] In one aspect of the analyzer system 300, the detector 309
of the analyzer system detects the photons emitted by the
fluorescent label. In one embodiment, the photon detector is a
photodiode. In a further embodiment, the detector is an avalanche
photodiode detector. In some embodiments, the photodiodes can be
silicon photodiodes with a wavelength detection of 190 nm and 1100
nm. When germanium photodiodes are used, the wavelength of light
detected is between 400 nm to 1700 nm. In other embodiments, when
an indium gallium arsenide photodiode is used, the wavelength of
light detected by the photodiode is between 800 nm and 2600 nm.
When lead sulfide photodiodes are used as detectors, the wavelength
of light detected is between 1000 nm and 3500 nm.
[0164] In some embodiments, the optics of the electromagnetic
radiation source 301 and the optics of the detector 309 are
arranged in a conventional optical arrangement. In such an
arrangement, the electromagnetic radiation source and the detector
are aligned on different focal planes. The arrangement of the laser
and the detector optics of the analyzer system as shown in FIGS. 1A
and 1B is that of a conventional optical arrangement.
[0165] In some embodiments, the optics of the electromagnetic
radiation source and the optics of the detector are arranged in a
confocal optical arrangement. In such an arrangement, the
electromagnetic radiation source 301 and the detector 309 are
aligned on the same focal plane. The confocal arrangement renders
the analyzer more robust because the electromagnetic radiation
source 301 and the detector optics 309 do not need to be realigned
if the analyzer system is moved. This arrangement also makes the
use of the analyzer more simplified because it eliminates the need
to realign the components of the analyzer system. The confocal
arrangement for the analyzer 300 (FIG. 1A) and the analyzer 355
(FIG. 1B) are shown in FIGS. 3A and 3B respectively. FIG. 3A shows
that the beam 311 from an electromagnetic radiation source 301 is
focused by the microscope objective 315 to form one interrogation
space 314 (FIG. 2A) within the capillary flow cell 313. A dichroic
mirror 316, which reflects laser light but passes fluorescent
light, is used to separate the fluorescent light from the laser
light. Filter 317 that is positioned in front of the detector
eliminates any non-fluorescent light at the detector. In some
embodiments, an analyzer system configured in a confocal
arrangement can comprise two or more interrogations spaces. Such a
method has been previously disclosed and is incorporated by
reference from previous U.S. patent application Ser. No.
11/048,660.
[0166] The laser can be a tunable dye laser, such as a helium-neon
laser. The laser can be set to emit a wavelength of 632.8 nm.
Alternatively, the wavelength of the laser can be set to emit a
wavelength of 543.5 nm or 1523 nm. Alternatively, the
electromagnetic laser can be an argon ion laser. In such an
embodiment, the argon ion laser can be operated as a continuous gas
laser at about 25 different wavelengths in the visible spectrum,
the wavelength set between 408.9 and 686.1 nm but at its optimum
performance set between 488 and 514.5 nm.
[0167] 1 Electromagnetic Radiation Source
[0168] In some embodiments of the analyzer system a
chemiluminescent label may be used. In such an embodiment, it may
not be necessary to utilize an EM source for detection of the
particle. In another embodiment, the extrinsic label or intrinsic
characteristic of the particle is a light-interacting label or
characteristic, such as a fluorescent label or a light-scattering
label. In such an embodiment, a source of EM radiation is used to
illuminate the label and/or the particle. EM radiation sources for
excitation of fluorescent labels are preferred.
[0169] In some embodiments, the analyzer system consists of an
electromagnetic radiation source 301. Any number of radiation
sources may be used in any one analyzer system 300 without
departing from the scope of the invention. Multiple sources of
electromagnetic radiation have been previously disclosed and are
incorporated by reference from previous U.S. patent application
Ser. No. 11/048,660. In some embodiments, all the continuous wave
electromagnetic (EM) radiation sources emit electromagnetic
radiation at the same wavelengths. In other embodiments, different
sources emit different wavelengths of EM radiation.
[0170] In one embodiment, the EM source(s) 301, 351, 352 are
continuous wave lasers producing wavelengths of between 200 nm and
1000 nm. Such EM sources have the advantage of being small, durable
and relatively inexpensive. In addition, they generally have the
capacity to generate larger fluorescent signals than other light
sources. Specific examples of suitable continuous wave EM sources
include, but are not limited to: lasers of the argon, krypton,
helium-neon, helium-cadmium types, as well as, tunable diode lasers
(red to infrared regions), each with the possibility of frequency
doubling. The lasers provide continuous illumination with no
accessory electronic or mechanical devices, such as shutters, to
interrupt their illumination. In an embodiment where a continuous
wave laser is used, an electromagnetic radiation source of 3 mW may
be of sufficient energy to excite a fluorescent label. A beam from
a continuous wave laser of such energy output may be between 2 to 5
.mu.m in diameter. The time of exposure of the particle to laser
beam in order to be exposed to 3 mW may be a time period of about 1
msec. In alternate embodiments, the time of exposure to the laser
beam may be equal to or less than about 500 .mu.sec. In an
alternate embodiment, the time of exposure may be equal to or less
than about 100 .mu.sec. In an alternate embodiment, the time of
exposure may be equal to or less than about 50 .mu.sec. In an
alternate embodiment, the time of exposure may be equal to or less
than about 10 .mu.sec.
[0171] LEDs are another low-cost, high reliability illumination
source. Recent advances in ultra-bright LEDs and dyes with high
absorption cross-section and quantum yield support the
applicability of LEDs to single particle detection. Such lasers
could be used alone or in combination with other light sources such
as mercury arc lamps, elemental arc lamps, halogen lamps, arc
discharges, plasma discharges, light-emitting diodes, or
combination of these.
[0172] In other embodiments, the EM source could be in the form of
a pulse wave laser. In such an embodiment, the pulse size of the
laser is an important factor. In such an embodiment, the size,
focus spot, and the total energy emitted by the laser is important
and must be of sufficient energy as to be able to excite the
fluorescent label. When a pulse laser is used, a pulse of longer
duration may be required. In some embodiments a laser pulse of 2
nanoseconds may be used. In some embodiments a laser pulse of 5
nanoseconds may be used. In some embodiments a pulse of between 2
to 5 nanoseconds may be used.
[0173] The optimal laser intensity depends on the photo bleaching
characteristics of the single dyes and the length of time required
to traverse the interrogation space (including the speed of the
particle, the distance between interrogation spaces if more than
one is used and the size of the interrogation space(s)). To obtain
a maximal signal, it is desirable to illuminate the sample at the
highest intensity which will not result in photo bleaching a high
percentage of the dyes. The preferred intensity is one such that no
more that 5% of the dyes are bleached by the time the particle has
traversed the interrogation space.
[0174] The power of the laser is set depending on the type of dye
molecules that need to be stimulated and the length of time the dye
molecules are stimulated, and/or the speed with which the dye
molecules pass through the capillary flow cell. Laser power is
defined as the rate at which energy is delivered by the beam and is
measured in units of Joules/second, or Watts. It will be
appreciated that the greater the power output of the laser, the
shorter the time that the laser illuminates the particle may be,
while providing a constant amount of energy to the interrogation
space while the particle is passing through the space. Thus, in
some embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is more than
about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, or 100 microJoule. In some
embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is less than
about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35,
40, 45, 50, 60, 70, 80, 90, 100, or 110 microJoule. In some
embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is between
about 0.1 and 100 microJoule. In some embodiments, the combination
of laser power and time of illumination is such that the total
energy received by the interrogation space during the time of
illumination is between about 1 and 100 microJoule. In some
embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is between
about 1 and 50 microJoule. In some embodiments, the combination of
laser power and time of illumination is such that the total energy
received by the interrogation space during the time of illumination
is between about 2 and 50 microJoule. In some embodiments, the
combination of laser power and time of illumination is such that
the total energy received by the interrogation space during the
time of illumination is between about 3 and 60 microJoule. In some
embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is between
about 3 and 50 microJoule. In some embodiments, the combination of
laser power and time of illumination is such that the total energy
received by the interrogation space during the time of illumination
is between about 3 and 40 microJoule. In some embodiments, the
combination of laser power and time of illumination is such that
the total energy received by the interrogation space during the
time of illumination is between about 3 and 30 microJoule. In some
embodiments, the combination of laser power and time of
illumination is such that the total energy received by the
interrogation space during the time of illumination is about 1
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 3
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 5
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 10
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 15
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 20
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 30
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 40
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 50
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 60
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 70
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 80
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 90
microJoule. In some embodiments, the combination of laser power and
time of illumination is such that the total energy received by the
interrogation space during the time of illumination is about 100
microJoule.
[0175] In some embodiments, the laser power output is set to at
least about 1 mW, 2 mW, 3 mW, 4 mW, 5 mW, 6, mw, 7 mW, 8 mW, 9 mW,
10 mW, 13 mW, 15 mW, 20 mW, 25 mW, 30 mW, 40 mW, 50 mW, 60 mW, 70
mW, 80 mW, 90 mW, 100 mW, or more than 100 mW. In some embodiments,
the laser power output is set to at least about 1 mW. In some
embodiments, the laser power output is set to at least about 3 mW.
In some embodiments, the laser power output is set to at least
about 5 mW. In some embodiments, the laser power output is set to
at least about 10 mW. In some embodiments, the laser power output
is set to at least about 20 mW. In some embodiments, the laser
power output is set to at least about 30 mW. In some embodiments,
the laser power output is set to at least about 40 mW. In some
embodiments, the laser power output is set to at least about 50 mW.
In some embodiments, the laser power output is set to at least
about 60 mW. In some embodiments, the laser power output is set to
at least about 90 mW.
[0176] The time that the laser illuminates the interrogation space
can be set to no less than about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40,
50, 60, 70, 80, 90, 100, 150, 200, 150, 300, 350, 400, 450, 500,
600, 700, 800, 900, or 1000 microseconds. The time that the laser
illuminates the interrogation space can be set to no more than
about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,
200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500,
or 2000 microseconds. The time that the laser illuminates the
interrogation space can be set between about 1 and 1000
microseconds. The time that the laser illuminates the interrogation
space can be set between about 5 and 500 microseconds. The time
that the laser illuminates the interrogation space can be set
between about 5 and 100 microseconds. The time that the laser
illuminates the interrogation space can be set between about 10 and
100 microseconds. The time that the laser illuminates the
interrogation space can be set between about 10 and 50
microseconds. The time that the laser illuminates the interrogation
space can be set between about 10 and 20 microseconds. The time
that the laser illuminates the interrogation space can be set
between about 5 and 50 microseconds. The time that the laser
illuminates the interrogation space can be set between about 1 and
100 microseconds. In some embodiments, the time that the laser
illuminates the interrogation space is about 1 microsecond. In some
embodiments, the time that the laser illuminates the interrogation
space is about 5 microseconds. In some embodiments, the time that
the laser illuminates the interrogation space is about 10
microseconds. In some embodiments, the time that the laser
illuminates the interrogation space is about 25 microseconds. In
some embodiments, the time that the laser illuminates the
interrogation space is about 50 microseconds. In some embodiments,
the time that the laser illuminates the interrogation space is
about 100 microseconds. In some embodiments, the time that the
laser illuminates the interrogation space is about 250
microseconds. In some embodiments, the time that the laser
illuminates the interrogation space is about 500 microseconds. In
some embodiments, the time that the laser illuminates the
interrogation space is about 1000 microseconds.
[0177] For example, the time that the laser illuminates the
interrogation space can be set to 1 millisecond, 250 microseconds,
100 microseconds, 50 microseconds, 25 microseconds or 10
microseconds with a laser that provides a power output of 3 mW, 4
mw, 5 mW, or more than 5 mW. In some embodiments, a label is
illuminated with a laser that provides a power output of 3 mW and
illuminates the label for about 1000 microseconds. In other
embodiments, a label is illuminated for less than 1000 milliseconds
with a laser providing a power output of not more than about 20 mW.
In other embodiments, the label is illuminated with a laser power
output of 20 mW for less than or equal to about 250 microseconds.
In some embodiments, the label is illuminated with a laser power
output of about 5 mW for less than or equal to about 1000
microseconds.
[0178] 2. Capillary Flow Cell
[0179] The capillary flow cell is fluidly connected to the sample
system. In one embodiment, the interrogation space 314 of an
analyzer system, is determined by the cross sectional area of the
corresponding beam 311 and by a segment of the beam within the
field of view of the detector 309. In one embodiment of the
analyzer system, the interrogation space 314 has a volume, as
defined herein, of between about between about 0.01 and 500 pL, or
between about 0.01 pL and 100 pL, or between about 0.01 pL and 10
pL, or between about 0.01 pL and 1 pL, or between about 0.01 pL and
0.5 pL, or between about 0.02 pL and about 300 pL, or between about
0.02 pL and about 50 pL or between about 0.02 pL and about 5 pL or
between about 0.02 pL and about 0.5 pL or between about 0.02 pL and
about 2 pL, or between about 0.05 pL and about 50 pL, or between
about 0.05 pL and about 5 pL, or between about 0.05 pL and about
0.5 pL, or between about 0.05 pL and about 0.2 pL, or between about
0.1 pL and about 25 pL. In some embodiments, the interrogation
space has a volume between about 0.01 pL and 10 pL. In some
embodiments, the interrogation space 314 has a volume between about
0.01 pL and 1 pL. In some embodiments, the interrogation space 314
has a volume between about 0.02 pL and about 5 pL. In some
embodiments, the interrogation space 314 has a volume between about
0.02 pL and about 0.5 pL. In some embodiments, the interrogation
space 314 has a volume between about 0.05 pL and about 0.2 pL. In
some embodiments, the interrogation space 314 has a volume of about
0.1 pL. Other useful interrogation space volumes are as described
herein. It should be understood by one skilled in the art that the
interrogation space 314 can be selected for maximum performance of
the analyzer. Although very small interrogation spaces have been
shown to minimize the background noise, large interrogation spaces
have the advantage that low concentration samples can be analyzed
in a reasonable amount of time. In embodiments in which two
interrogation spaces 370 and 371 are used, volumes such as those
described herein for a single interrogation space 314 may be
used.
[0180] In one embodiment of the present invention, the
interrogation spaces are large enough to allow for detection of
particles at concentrations ranging from about 1000 femtomolar (fM)
to about 1 zeptomolar (zM). In one embodiment of the present
invention, the interrogation spaces are large enough to allow for
detection of particles at concentrations ranging from about 1000 fM
to about 1 attomolar (aM). In one embodiment of the present
invention, the interrogation spaces are large enough to allow for
detection of particles at concentrations ranging from about 10 fM
to about 1 attomolar (aM). In many cases, the large interrogation
spaces allow for the detection of particles at concentrations of
less than about 1 fM without additional pre-concentration devices
or techniques. One skilled in the art will recognize that the most
appropriate interrogation space size depends on the brightness of
the particles to be detected, the level of background signal, and
the concentration of the sample to be analyzed.
[0181] The size of the interrogation space 314 can be limited by
adjusting the optics of the analyzer. In one embodiment, the
diameter of the beam 311 can be adjusted to vary the volume of the
interrogation space 314. In another embodiment, the field of view
of the detector 309 can be varied. Thus, the source 301 and the
detector 309 can be adjusted so that single particles will be
illuminated and detected within the interrogation space 314. In
another embodiment, the width of aperture 306 (FIG. 1A) that
determine the field of view of the detector 309 is variable. This
configuration allows for altering the interrogation space, in near
real time, to compensate for more or less concentrated samples,
ensuring a low probability of two or more particles simultaneously
being within an interrogation space. Similar alterations for two or
more interrogation spaces, 370 and 371, may performed.
[0182] In another embodiment, the interrogation space can be
defined through the use of a calibration sample of known
concentration that is passed through the capillary flow cell prior
to the actual sample being tested. When only one single particle is
detected at a time in the calibration sample as the sample is
passing through the capillary flow cell, the depth of focus
together with the diameter of the beam of the electromagnetic
radiation source determines the size of the interrogation space in
the capillary flow cell.
[0183] Physical constraints to the interrogation spaces can also be
provided by a solid wall. In one embodiment, the wall is one or
more of the walls of a flow cell 313 (FIG. 2A), when the sample
fluid is contained within a capillary. In one embodiment, the cell
is made of glass, but other substances transparent to light in the
range of about 200 to about 1,000 nm or higher, such as quartz,
fused silica, and organic materials such as Teflon, nylon,
plastics, such as polyvinylchloride, polystyrene, and polyethylene,
or any combination thereof, may be used without departing from the
scope of the present invention. Although other cross-sectional
shapes (e.g., rectangular, cylindrical) may be used without
departing from the scope of the present invention, in one
embodiment the capillary flow cell 313 has a square cross section.
In another embodiment, the interrogation space may be defined at
least in part by a channel (not shown) etched into a chip (not
shown). Similar considerations apply to embodiments in which two
interrogation spaces are used (370 and 371 in FIG. 2B).
[0184] The interrogation space is bathed in a fluid. In one
embodiment, the fluid is aqueous. In other embodiments, the fluid
is non-aqueous or a combination of aqueous and non-aqueous fluids.
In addition the fluid may contain agents to adjust pH, ionic
composition, or sieving agents, such as soluble macroparticles or
polymers or gels. It is contemplated that valves or other devices
may be present between the interrogation spaces to temporarily
disrupt the fluid connection. Interrogation spaces temporarily
disrupted are considered to be connected by fluid.
[0185] In another embodiment of the invention, an interrogation
space is the single interrogation space present within the flow
cell 313 which is constrained by the size of a laminar flow of the
sample material within a diluent volume, also called sheath flow.
In these and other embodiments, the interrogation space can be
defined by sheath flow alone or in combination with the dimensions
of the illumination source or the field of view of the detector.
Sheath flow can be configured in numerous ways, including: The
sample material is the interior material in a concentric laminar
flow, with the diluent volume in the exterior; the diluent volume
is on one side of the sample volume; the diluent volume is on two
sides of the sample material; the diluent volume is on multiple
sides of the sample material, but not enclosing the sample material
completely; the diluent volume completely surrounds the sample
material; the diluent volume completely surrounds the sample
material concentrically; the sample material is the interior
material in a discontinuous series of drops and the diluent volume
completely surrounds each drop of sample material.
[0186] In some embodiments, single molecule detectors of the
invention comprise no more than one interrogation space. In some
embodiments, multiple interrogation spaces are used. Multiple
interrogation spaces have been previously disclosed and are
incorporated by reference from U.S. patent application Ser. No.
11/048,660. One skilled in the art will recognize that in some
cases the analyzer will contain 2, 3, 4, 5, 6 or more distinct
interrogation spaces.
[0187] 3. Motive Force
[0188] In one embodiment of the analyzer system, the particles are
moved through the interrogation space by a motive force. In some
embodiments, the motive force for moving particles is pressure. In
some embodiments, the pressure is supplied by a pump, and air
pressure source, a vacuum source, a centrifuge, or a combination
thereof. In some embodiments, the motive force for moving particles
is an electrokinetic force. The use of an electrokinetic force as a
motive force has been previously disclosed in a prior application
and is incorporated by reference from U.S. patent application Ser.
No. 11/048,660.
[0189] In one embodiment, pressure can be used as a motive force to
move particles through the interrogation space of the capillary
flow cell. In a further embodiment, pressure is supplied to move
the sample by means of a pump. Suitable pumps are known in the art.
In one embodiment, pumps manufactured for HPLC applications, such
as those made by Scivax, Inc. can be used as a motive force. In
other embodiments, pumps manufactured for microfluidics
applications can be used when smaller volumes of sample are being
pumped. Such pumps are described in U.S. Pat. Nos. 5,094,594,
5,730,187, 6,033,628, and 6,533,553, which discloses devices which
can pump fluid volumes in the nanoliter or picoliter range.
Preferably all materials within the pump that come into contact
with sample are made of highly inert materials, e.g.,
polyetheretherketone (PEEK), fused silica, or sapphire.
[0190] A motive force is necessary to move the sample through the
capillary flow cell to push the sample through the interrogation
space for analysis. A motive force is also required to push a
flushing sample through the capillary flow cell after the sample
has been passed through. A motive force is also required to push
the sample back out into a sample recovery vessel, when sample
recovery is employed. Standard pumps come in a variety of sizes,
and the proper size may be chosen to suit the anticipated sample
size and flow requirements. In some embodiments, separate pumps are
used for sample analysis and for flushing of the system. The
analysis pump may have a capacity of approximately 0.000001 mL to
approximately 10 mL, or approximately 0.001 mL to approximately 1
mL, or approximately 0.01 mL to approximately 0.2 mL, or
approximately 0.005, 0.01, 0.05, 0.1, or 0.5 mL. Flush pumps may be
of larger capacity than analysis pumps. Flush pumps may have a
volume of about 0.01 mL to about 20 mL, or about 0.1 mL to about 10
mL, or about 0.1 mL to about 2 mL, or about or about 0.05, 0.1,
0.5, 1, 5, or 10 mL. These pump sizes are illustrative only, and
those of skill in the art will appreciate that the pump size may be
chosen according to the application, sample size, viscosity of
fluid to be pumped, tubing dimensions, rate of flow, temperature,
and other factors well known in the art. In some embodiments, pumps
of the system are driven by stepper motors, which are easy to
control very accurately with a microprocessor.
[0191] In preferred embodiments, the flush and analysis pumps are
used in series, with special check valves to control the direction
of flow. The plumbing is designed so that when the analysis pump
draws up the maximum sample, the sample does not reach the pump
itself. This is accomplished by choosing the ID and length of the
tubing between the analysis pump and the analysis capillary such
that the tubing volume is greater than the stroke volume of the
analysis pump.
[0192] 4. Detectors
[0193] In one embodiment, light (e.g., light in the ultra-violet,
visible or infrared range) emitted by a fluorescent label after
exposure to electromagnetic radiation is detected. The detector 309
(FIG. 1A), or detectors (364, 365, FIG. 1B), is capable of
capturing the amplitude and duration of photon bursts from a
fluorescent label-moiety complex, and further converting the
amplitude and duration of the photon burst to electrical signals.
Detection devices such as CCD cameras, video input module cameras,
and Streak cameras can be used to produce images with contiguous
signals. In another embodiment, devices such as a bolometer, a
photodiode, a photodiode array, avalanche photodiodes, and
photomultipliers which produce sequential signals may be used. Any
combination of the aforementioned detectors may also be used. In
one embodiment, avalanche photodiodes are used for detecting
photons.
[0194] Using specific optics between an interrogation space 314
(FIG. 2A) and its corresponding detector 309 (FIG. 1A), several
distinct characteristics of the emitted electromagnetic radiation
can be detected including: emission wavelength, emission intensity,
burst size, burst duration, and fluorescence polarization. In some
embodiments, the detector 309 is a photodiode that is used in
reverse bias. A photodiode set in reverse bias usually has an
extremely high resistance. This resistance is reduced when light of
an appropriate frequency shines on the P/N junction. Hence, a
reverse biased diode can be used as a detector by monitoring the
current running through it. Circuits based on this effect are more
sensitive to light than ones based on zero bias.
[0195] In one embodiment of the analyzer system, the photodiode can
be an avalanche photodiode, which can be operated with much higher
reverse bias than conventional photodiodes, thus allowing each
photo-generated carrier to be multiplied by avalanche breakdown,
resulting in internal gain within the photodiode, which increases
the effective responsiveness (sensitivity) of the device. The
choice of photodiode is determined by the energy or emission
wavelength emitted by the fluorescently labeled particle. In some
embodiments, the photodiode is a silicon photodiode that detects
energy in the range of 190-1100 nm; in another embodiment the
photodiode is a germanium photodiode that detects energy in the
range of 800-1700 nm; in another embodiment the photodiode is an
indium gallium arsenide photodiode that detects energy in the range
of 800-2600 nm; and in yet other embodiments, the photodiode is a
lead sulfide photodiode that detects energy in the range of between
less than 1000 nm to 3500 nm. In some embodiments, the avalanche
photodiode is a single-photon detector designed to detect energy in
the 400 nm to 1100 nm wavelength range. Single photon detectors are
commercially available (for example Perkin Elmer, Wellesley,
Mass.).
[0196] In some embodiments the detector is a avalanche photodiode
detector that detects energy between 300 nm and 1700 nm. In one
embodiment, silicon avalanche photodiodes can be used to detect
wavelengths between 300 nm and 1100 nm. Indium gallium arsenic
photodiodes can be used to detect wavelengths between 900 nm and
1700 nm. In some embodiments, an analyzer system can comprise at
least one detector; in other embodiments, the analyzer system can
comprise at least two detectors, and each detector can be chosen
and configured to detect light energy at a specific wavelength
range. For example, two separate detectors can be used to detect
particles that have been tagged with different labels, which upon
excitation with an EM source, will emit photons with energy in
different spectra. In one embodiment, an analyzer system can
comprise a first detector that can detect fluorescent energy in the
range of 450-700 nm such as that emitted by a green dye (e.g. Alexa
546); and a second detector that can detect fluorescent energy in
the range of 620-780 nm such as that emitted by a far-red dye (e.g.
Alexa 647). Detectors for detecting fluorescent energy in the range
of 400-600 nm such as that emitted by blue dyes (e.g. Hoechst
33342), and for detecting energy in the range of 560-700 nm such as
that emitted by red dyes (Alexa 546 and Cy3) can also be used.
[0197] A system comprising two or more detectors can be used to
detect individual particles that are each tagged with two or more
labels that emit light in different spectra. For example, two
different detectors can detect an antibody that has been tagged
with two different dye labels. Alternatively, an analyzer system
comprising two detectors can be used to detect particles of
different types, each type being tagged with a different dye
molecules, or with a mixture of two or more dye molecules. For
example, two different detectors can be used to detect two
different types of antibodies that recognize two different
proteins, each type being tagged with a different dye label or with
a mixture of two or more dye label molecules. By varying the
proportion of the two or more dye label molecules, two or more
different particle types can be individually detected using two
detectors. It is understood that three or more detectors can be
used without departing from the scope of the invention.
[0198] It should be understood by one skilled in the art that one
or more detectors can be configured at each interrogation space,
whether one or more interrogation spaces are defined within a flow
cell, and that each detector may be configured to detect any of the
characteristics of the emitted electromagnetic radiation listed
above. The use of multiple detectors, e.g., for multiple
interrogation spaces, has been previously disclosed in a prior
application and is incorporated by reference here from U.S. patent
application Ser. No. 11/048,660. Once a particle is labeled to
render it detectable (or if the particle possesses an intrinsic
characteristic rendering it detectable), any suitable detection
mechanism known in the art may be used without departing from the
scope of the present invention, for example a CCD camera, a video
input module camera, a Streak camera, a bolometer, a photodiode, a
photodiode array, avalanche photodiodes, and photomultipliers
producing sequential signals, and combinations thereof. Different
characteristics of the electromagnetic radiation may be detected
including: emission wavelength, emission intensity, burst size,
burst duration, fluorescence polarization, and any combination
thereof.
[0199] C. Sampling System
[0200] In a further embodiment, the analyzer system may include a
sampling system to prepare the sample for introduction into the
analyzer system. The sampling system included is capable of
automatically sampling a plurality of samples and providing a fluid
communication between a sample container and a first interrogation
space.
[0201] In some embodiments, the analyzer system of the invention
includes a sampling system for introducing an aliquot of a sample
into the single particle analyzer for analysis. Any mechanism that
can introduce a sample may be used. Samples can be drawn up using
either a vacuum suction created by a pump or by pressure applied to
the sample that would push liquid into the tube, or by any other
mechanism that serves to introduce the sample into the sampling
tube. Generally, but not necessarily, the sampling system
introduces a sample of known sample volume into the single particle
analyzer; in some embodiments where the presence or absence of a
particle or particles is detected, precise knowledge of the sample
size is not critical. In preferred embodiments the sampling system
provides automated sampling for a single sample or a plurality of
samples. In embodiments where a sample of known volume is
introduced into the system, the sampling system provides a sample
for analysis of more than about 0.0001, 0.001, 0.01, 0.1, 1, 2, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500,
or 2000 .mu.l. In some embodiments the sampling system provides a
sample for analysis of less than about 2000, 1000, 500, 200, 100,
90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, or 0.001
.mu.l. In some embodiments the sampling system provides a sample
for analysis of between about 0.01 and 1500 .mu.l, or about 0.1 and
1000 .mu.l, or about 1 and 500 .mu.l, or about 1 and 100 .mu.l, or
about 1 and 501, or about 1 and 20 .mu.l. In some embodiments, the
sampling system provides a sample for analysis between about 5
.mu.l and 200 .mu.l, or about 5 .mu.l and about 100 .mu.l, or about
5 .mu.l and 50 .mu.l. In some embodiments, the sampling system
provides a sample for analysis between about 10 .mu.l and 200
.mu.l, or between about 10 .mu.l and 100 ul, or between about 10
.mu.l and 50 .mu.l. In some embodiments, the sampling system
provides a sample for analysis between about 0.5 .mu.l and about 50
.mu.l.
[0202] In some embodiments, the sampling system provides a sample
size that can be varied from sample to sample. In these
embodiments, the sample size may be any one of the sample sizes
described herein, and may be changed with every sample, or with
sets of samples, as desired.
[0203] Sample volume accuracy, and sample to sample volume
precision of the sampling system, is required for the analysis at
hand. In some embodiments, the precision of the sampling volume is
determined by the pumps used, typically represented by a CV of less
than about 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or
0.01% of sample volume. In some embodiments, the sample to sample
precision of the sampling system is represented by a CV of less
than about 50, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or
0.01%. In some embodiments, the intra-assay precision of the
sampling system is represented by a CV of less than about 10, 5, 1,
0.5, or 0.1%. In some embodiments, the intra-assay precision of the
sampling system shows a CV of less than about 5%. In some
embodiments, the interassay precision of the sampling system is
represented by a CV of less than about 10, 5, or 1%. In some
embodiments, the interassay precision of the sampling system shows
a CV of less than about 5%.
[0204] In some embodiments, the sampling system provides low sample
carryover, advantageous in that an additional wash step is not
required between samples. Thus, in some embodiments, sample
carryover is less than about 1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02,
0.01, 0.005, or 0.001%. In some embodiments, sample carryover is
less than about 0.02%. In some embodiments, sample carryover is
less than about 0.01%.
[0205] In some embodiments the sampler provides a sample loop. In
these embodiments, multiple samples are drawn into tubing
sequentially and each is separated from the others by a "plug" of
buffer. The samples typically are read one after the other with no
flushing in between. Flushing is done once at the end of the loop.
In embodiments where a buffer "plug" is used, the plug may be
recovered ejecting the buffer plug into a separate well of a
microtiter plate.
[0206] The sampling system may be adapted for use with standard
assay equipment, for example, a 96-well microtiter plate, or,
preferably, a 384-well plate. In some embodiments the system
includes a 96 well plate positioner and a mechanism to dip the
sample tube into and out of the wells, e.g., a mechanism providing
movement along the X, Y, and Z axes. In some embodiments, the
sampling system provides multiple sampling tubes from which samples
may be stored and extracted from, when testing is commenced. In
some embodiments, all samples from the multiple tubes are analyzed
on one detector. In other embodiments, multiple single molecule
detectors may be connected to the sample tubes. Samples may be
prepared by steps that include operations performed on sample in
the wells of the plate prior to sampling by the sampling system, or
sample may be prepared within the analyzer system, or some
combination of both.
[0207] D. Sample Preparation System
[0208] Sample preparation includes the steps necessary to prepare a
raw sample for analysis. These steps can involve, by way of
example, one or more steps of: separation steps such as
centrifugation, filtration, distillation, chromatography;
concentration, cell lysis, alteration of pH, addition of buffer,
addition of diluents, addition of reagents, heating or cooling,
addition of label, binding of label, cross-linking with
illumination, separation of unbound label, inactivation and/or
removal of interfering compounds and any other steps necessary for
the sample to be prepared for analysis by the single particle
analyzer. In some embodiments, blood is treated to separate out
plasma or serum. Additional labeling, removal of unbound label,
and/or dilution steps may also be performed on the serum or plasma
sample.
[0209] In some embodiments, the analyzer system includes a sample
preparation system that performs some or all of the processes
needed to provide a sample ready for analysis by the single
particle analyzer. This system may perform any or all of the steps
listed above for sample preparation. In some embodiments samples
are partially processed by the sample preparation system of the
analyzer system. Thus, in some embodiments, a sample may be
partially processed outside the analyzer system first. For example,
the sample may be centrifuged first. The sample may then be
partially processed inside the analyzer by a sample preparation
system. Processing inside the analyzer includes labeling the
sample, mixing the sample with a buffer and other processing steps
that will be known to one in the art. In some embodiments, a blood
sample is processed outside the analyzer system to provide a serum
or plasma sample, which is introduced into the analyzer system and
further processed by a sample preparation system to label the
particle or particles of interest and, optionally, to remove
unbound label. In other embodiments preparation of the sample can
include immunodepletion of the sample to remove particles that are
not of interest or to remove particles that can interfere with
sample analysis. In yet other embodiments, the sample can be
depleted of particles that can interfere with the analysis of the
sample. For example, sample preparation can include the depletion
of heterophilic antibodies, which are known to interfere with
immunoassays that use non-human antibodies to directly or
indirectly detect a particle of interest. Similarly, other proteins
that interfere with measurements of the particles of interest can
be removed from the sample using antibodies that recognize the
interfering proteins.
[0210] In some embodiments, the sample can be subjected to solid
phase extraction prior to being assayed and analyzed. For example,
a serum sample that is assayed for cAMP can first be subjected to
solid phase extraction using a c18 column to which it binds. Other
proteins such as proteases, lipases and phosphatases are washed
from the column, and the cAMP is eluted essentially free of
proteins that can degrade or interfere with measurements of cAMP.
Solid phase extraction can be used to remove the basic matrix of a
sample, which can diminish the sensitivity of the assay. In yet
other embodiments, the particles of interest present in a sample
may be concentrated by drying or lyophilizing a sample and
solubilizing the particles in a smaller volume than that of the
original sample. For example, a sample of exhaled breath condensate
(EBC) can be dried and resuspended in a small volume of a suitable
buffer solution to enhance the detection of the particle of
interest.
[0211] In some embodiments the analyzer system provides a sample
preparation system that provides complete preparation of the sample
to be analyzed on the system, such as complete preparation of a
blood sample, a saliva sample, a urine sample, a cerebrospinal
fluid sample, a lymph sample, a BAL sample, an exhaled breath
condensate sample (EBC), a biopsy sample, a forensic sample, a
bioterrorism sample, and the like. In some embodiments the analyzer
system provides a sample preparation system that provides some or
all of the sample preparation. In some embodiments, the initial
sample is a blood sample that is further processed by the analyzer
system. In some embodiments, the sample is a serum or plasma sample
that is further processed by the analyzer system. The serum or
plasma sample may be further processed by, e.g., contacting with a
label that binds to a particle or particles of interest; the sample
may then be used with or without removal of unbound label.
[0212] In some embodiments, sample preparation is performed, either
outside the analysis system or in the sample preparation component
of the analysis system, on one or more microtiter plates, such as a
96-well plate. Reservoirs of reagents, buffers, and the like can be
in intermittent fluid communication with the wells of the plate by
means of tubing or other appropriate structures, as are well-known
in the art. Samples may be prepared separately in 96 well plates or
tubes. Sample isolation, label binding and, if necessary, label
separation steps may be done on one plate. In some embodiments,
prepared particles are then released from the plate and samples are
moved into tubes for sampling into the sample analysis system. In
some embodiments, all steps of the preparation of the sample are
done on one plate and the analysis system acquires sample directly
from the plate. Although this embodiment is described in terms of a
96-well plate, it will be appreciated that any vessel for
containing one or more samples and suitable for preparation of
sample may be used. For example, standard microtiter plates of 384
or 1536 wells may be used. More generally, in some embodiments, the
sample preparation system is capable of holding and preparing more
than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,
500, 1000, 5000, or 10,000 samples. In some embodiments, multiple
samples may be sampled for analysis in multiple analyzer systems.
Thus, in some embodiments, 2 samples, or more than about 2, 3, 4,
5, 7, 10, 15 20, 50, or 100 samples are sampled from the sample
preparation system and run in parallel on multiple sample analyzer
systems.
[0213] Microfluidics systems may also be used for sample
preparation and as sample preparation systems that are part of
analyzer systems, especially for samples suspected of containing
concentrations of particles high enough that detection requires
smaller samples. Principles and techniques of microfluidic
manipulation are known in the art. See, e.g., U.S. Pat. Nos.
4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,658,413;
5,653,939; 5,653,859; 5,645,702; 5,605,662; 5,571,410; 5,543,838;
5,480,614, 5,716,825; 5,603,351; 5,858,195; 5,863,801; 5,955,028;
5,989,402; 6,041,515; 6,071,478; 6,355,420; 6,495,104; 6,386,219;
6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744; 6,361,671;
6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992; 6,537,437;
6,599,436; 6,811,668 and published PCT patent application no.
WO9955461(A1). Samples may be prepared in series or in parallel,
for use in a single or multiple analyzer systems.
[0214] Preferably, the sample comprises a buffer. The buffer may be
mixed with the sample outside the analyzer system, or it may be
provided by the sample preparation mechanism. While any suitable
buffer can be used, the preferable buffer has low fluorescence
background, is inert to the detectably labeled particle, can
maintain the working pH and, in embodiments wherein the motive
force is electrokinetic, has suitable ionic strength for
electrophoresis. The buffer concentration can be any suitable
concentration, such as in the range from about 1 to about 200 mM.
Any buffer system may be used as long as it provides for
solubility, function, and delectability of the molecules of
interest. Preferably, for application using pumping, the buffer is
selected from the group consisting of phosphate, glycine, acetate,
citrate, acidulate, carbonate/bicarbonate, imidazole,
triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces,
PIPES, MOPSO, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS,
TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine,
HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS.
The buffer can also be selected from the group consisting of
Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid (MES),
4-morpholine propanesulfonic acid (MOPS) and
2-amino-2-methyl-1-propanol hydrochloride (AMP). A useful buffer is
2 mM Tris/borate at pH 8.1, but Tris/glycine and Tris/HCl are also
acceptable. Other buffers are as described herein.
[0215] Buffers useful for electrophoresis are disclosed in a prior
application and are incorporated by reference herein from U.S.
patent application Ser. No. 11/048,660.
[0216] E. Sample Recovery
[0217] One highly useful feature of embodiments of the analyzers
and analysis systems of the invention is that the sample can be
analyzed without consuming it. This can be especially important
when sample materials are limited. Recovering the sample also
allows one to do other analyses or reanalyze it. The advantages of
this feature for applications where sample size is limited and/or
where the ability to reanalyze the sample is desirable, e.g.,
forensic, drug screening, and clinical diagnostic applications,
will be apparent to those of skill in the art.
[0218] Thus, in some embodiments, the analyzer system of the
invention further provides a sample recovery system for sample
recovery after analysis. In these embodiments, the system includes
mechanisms and methods by which the sample is drawn into the
analyzer, analyzed and then returned, e.g., by the same path, to
the sample holder, e.g., the sample tube. Because no sample is
destroyed and because it does not enter any of the valves or other
tubing, it remains uncontaminated. In addition, because all the
materials in the sample path are highly inert, e.g., PEEK, fused
silica, or sapphire, there is little contamination from the sample
path. The use of the stepper motor controlled pumps (particularly
the analysis pump) allows precise control of the volumes drawn up
and pushed back out. This allows complete or nearly complete
recovery of the sample with little if any dilution by the flush
buffer. Thus, in some embodiments, more than about 50%, 60%, 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the
sample is recovered after analysis. In some embodiments, the
recovered sample is undiluted. In some embodiments, the recovered
sample is diluted less than about 1.5-fold, 1.4-fold, 1.3-fold,
1.2-fold, 1.1-fold, 1.05-fold, 1.01-fold, 1.005-fold, or
1.001-fold.
[0219] For sampling and/or sample recovery, any mechanism for
transporting a liquid sample from a sample vessel to the analyzer
may be used. In some embodiments the inlet end of the analysis
capillary has attached a short length of tubing, e.g., PEEK tubing
that can be dipped into a sample container, e.g. a test tube or
sample well, or can be held above a waste container. When flushing,
to clean the previous sample from the apparatus, this tube is
positioned above the waste container to catch the flush waste. When
drawing a sample in, the tube is put into the sample well or test
tube. Typically the sample is drawn in quickly, and then pushed out
slowly while observing particles within the sample. Alternatively,
in some embodiments, the sample is drawn in slowly during at least
part of the draw-in cycle; the sample may be analyzed while being
slowly drawn in. This can be followed by a quick return of the
sample and a quick flush. In some embodiments, the sample may be
analyzed both on the inward (draw-in) and outward (pull out) cycle,
which improves counting statistics, e.g., of small and dilute
samples, as well as confirming results, and the like. If it is
desired to save the sample, it can be pushed back out into the same
sample well it came from, or to another. If saving the sample is
not desired, the tubing is positioned over the waste container.
VI. Methods Using Highly Sensitive Analysis of Cardiac Troponin
[0220] The methods of the present invention make possible
measurement of cardiac troponin levels at concentrations far lower
than previously measured. Although cardiac troponin is an accepted
marker for cardiac muscle damage, its usefulness has been limited
by the fact that, with current methods of analysis, it is only
detectable after considerable damage to cardiac muscle has
occurred, because of the lack of sensitivity of current methods.
The Joint European Society of Cardiology/American College of
Cardiology committee for the Redefinition of Myocardial Infarction
has recommended that an increased concentration of cardiac troponin
be defined as a measurement exceeding the 99.sup.th percentile of
the distribution of cardiac troponin concentrations in the
reference group, a very low threshold. A total imprecision (CV) at
this decision limit of <10% is recommended. However, the
analytical imprecision obtained with presently available
immunoassays for cardiac troponins is not uniform, mainly at the
low concentration range. In addition, the assays that are currently
available lack sufficient sensitivity for detecting troponin levels
in nonclinical (normal) subjects, and a true baseline or a level of
troponin defined in a normal population, has not been defined. The
analyzer systems of the invention have been shown to be able to
consistently detect levels of cTnI at concentrations of less than
10 pg/ml with a total imprecision of less than 10% (See Examples).
Thus, the invention provides methods for diagnosis, prognosis, or
methods of treatment based on the highly sensitive detection of
cardiac troponin in individuals.
[0221] In some embodiments, the invention provides a method for
determining a diagnosis, prognosis, or method of treatment in an
individual by i) determining a concentration of cardiac troponin in
a sample or determining the concentrations of cardiac troponin in a
series of samples from the individual, where the concentration is
determined by a cardiac troponin assay with a limit of detection
for the cardiac troponin in said sample of less than about 50, 40,
30, 10, 5, 4, 3, 2 or 1 pg/ml, e.g., less than about 20 pg/ml; and
ii) determining a diagnosis, prognosis, or method of treatment in
said individual, based on the concentration in the sample, or on
the concentrations in the series of samples. The method of
determining the concentration of cardiac troponin includes any
suitable method with the requisite sensitivity, e.g., the methods
descried herein. In some embodiments, the methods utilize a method
of determining a concentration of cardiac troponin in the sample
where the method comprises detecting single molecules of troponin,
or complexes or fragments thereof.
[0222] In some embodiments, the threshold concentration of troponin
is determined by analyzing samples, e.g., blood, serum, or plasma
samples, from an apparently healthy population for cardiac
troponin, e.g., cardiac troponin I, and determining the level at
which 80, 90, 95, 96, 97, 98, 99, 99.5, or 99.9% of the population
fall below that level (concentration). This value is the threshold
value. In some embodiments, the threshold value is set at the
99.sup.th percentile. In some embodiments, the analyzing is
performed using a method with a level of detection for the cardiac
troponin of less than about 50, 20, 10, 5, or 1 pg/ml, e.g., less
than about 5 pg/ml.
[0223] In some embodiments, the invention provides a method for
determining a diagnosis, prognosis, or method of treatment in an
individual by comparing a value for a concentration of cardiac
troponin in a sample from the individual with a normal value or a
range of normal values for cardiac troponin, where the normal vale
or range of normal values is determined by a cardiac troponin assay
with a limit of detection for the cardiac troponin in said sample
of less than about 50, 40, 30, 10, 5, 4, 3, 2 or 1 pg/ml, e.g.,
less than about 20 pg/ml; and ii) determining a diagnosis,
prognosis, or method of treatment in said individual, based on
comparison.
[0224] In some embodiments, the cardiac troponin is cardiac
troponin I or cardiac troponin T. In some embodiments, the cardiac
troponin is cardiac troponin T. In some embodiments, the cardiac
troponin is cardiac troponin I. The method may use total troponin,
e.g., total cTnI, or cTnT, or total cTnI+cTnT, as described herein,
in determining a diagnosis, prognosis, or method of treatment. In
some embodiments, the method may use the concentration of free,
complexed, or fragments of the cardiac troponin, or a comparison of
these (e.g., a ratio), to determine a diagnosis, prognosis, or
method of treatment.
[0225] A. Samples
[0226] The sample or series of samples may be any suitable sample;
in some embodiments, the sample(s) will be blood, serum, or plasma.
In some embodiments, the sample or series of samples are serum
samples. The individual may be an animal, e.g., mammal, e.g.,
human.
[0227] A single sample may be taken, or a series of samples may be
taken. If a series of samples is taken, they may be taken at any
suitable interval, e.g., intervals of minutes, hours, days, weeks,
months, or years. In an acute clinical setting, typically a series
of samples will be taken over the course of hours and days, with
the samples separated by a matter of hours. When an individual is
followed for longer periods, sample intervals may be months or
years. Diagnosis, prognosis, or method of treatment may be
determined from a single sample, or from one or more of a series of
samples, or from changes in the series of samples, e.g., an
increase in concentration at a certain rate may indicate a severe
condition whereas increase at a slower rate or no increase may
indicate a relatively benign or less serious condition. The rate of
change may be measured over the course of hours, days, weeks,
months, or years. Rate of change in a given individual may, in some
cases, be more relevant than an absolute value. In acute setting,
an extremely rapid rate of change, e.g., a "spike", can indicate an
imminent, ongoing, or recent cardiac event. In other settings, a
rise in values over a period of days, weeks, months or years in an
individual can indicate ongoing and worsening cardiac damage, e.g.,
cardiac damage due to a cardiac condition (e.g., cardiac
hypertrophy or congestive heart failure) or cardiac damage due to a
non-cardiac condition (e.g., toxicity from drug exposure).
[0228] In some embodiments, at least one sample is taken during or
after a cardiac stress test. E.g., a sample may be taken before the
stress test, and one or more samples taken during the test.
Deviations in cardiac troponin levels between the sample before the
test and the sample(s) taken during the test can provide diagnostic
or prognostic information, e.g., indicate the likelihood of
coronary artery disease or other pathology associated with the
cardiac muscle. Other comparisons may be done as well, such as
comparisons of any of the samples to normal or threshold levels, or
determination of a rate of change in the concentration of cardiac
troponin in the samples, all of which may yield useful information
regarding cardiac and cardiovascular health, as well as other
conditions as described herein.
[0229] In some embodiments, at least one sample is taken at or near
the time the individual presents to a health professional with one
or more symptoms indicative of a condition that may involve cardiac
damage. Settings in which an individual may present to a health
care professional include, but are not limited to ambulatory,
urgent care, critical care, intensive care, monitoring unit,
inpatient, outpatient, physician office, medical clinic, emergency
response setting, including an ambulance, and health screening
settings. In some embodiments, one or more samples are taken from
the individual and are assayed for cardiac troponin locally, i.e.,
at or near the setting at which the samples are taken. For example,
an individual who presents at a hospital may have one or more
samples taken that are assayed for cardiac troponin within the
hospital. For example, an individual who presents at a hospital may
have one or more samples taken that are assayed for cardiac
troponin within the hospital. In some embodiments, one or more
samples are taken from the individual and are assayed for cardiac
troponin in a CLIA laboratory. In some embodiments, the individual
displays one or more symptoms consistent with acute coronary
syndrome. In some embodiments, the individual displays one or more
symptoms consistent with AMI. Such symptoms include, but are not
limited to, chest pain, chest pressure, arm pain, abnormal EKG,
abnormal enzyme levels, and shortness of breath.
[0230] B. Determination of Diagnosis, Prognosis, or Method of
Treatment
[0231] In some embodiments, step ii) includes comparing said
concentration or series of concentrations to a normal value for
said concentration, comparing said concentration or series of
concentrations to a predetermined threshold level, comparing said
concentration or series of concentrations to a baseline value, or
determining a rate of change of concentration for said series of
concentrations.
[0232] In some embodiments, step ii) comprises comparing said
concentration of troponin in said sample with a predetermined
threshold concentration, and determining a diagnosis, prognosis, or
method of treatment if the sample concentration is greater than the
threshold level. The threshold concentration can be determined by,
e.g., determining the 99th percentile concentration of troponin in
a group of individuals, and setting said threshold concentration at
said 99th percentile concentration. An example of this is given in
Examples.
[0233] Normal values, threshold values, rates of change, ratios of
values, and other useful diagnostic and prognostic indicators may
be established by methods well-known in the art. For example, these
values may be determined by comparing samples from a case
population and a control population, where the case population
exhibits the biological state for which diagnosis, prognosis, or
method of treatment is desired, and the control population does not
exhibit the biological state. In some embodiments, a longitudinal
study may be done, e.g., the case population may be a subset of the
control population that, over time, exhibits the biological state.
It will be appreciated that data from a plurality of studies may be
used to determine a consensus value or range of values for normal,
and for prognostic or diagnostic levels.
[0234] In developing diagnostic or prognostic test, data for one or
more potential markers may be obtained from a group of subjects.
The group of subjects is divided into at least two sets, and
preferably the first set and the second set each have an
approximately equal number of subjects. The first set includes
subjects who have been confirmed as having a disease or, more
generally, being in a first condition state. For example, this
first set of patients may be those that have recently had a disease
incidence, or may be those having a specific type of disease, such
as AMI. The confirmation of the condition state may be made through
a more rigorous and/or expensive testing such as MRI or CT.
Hereinafter, subjects in this first set will be referred to as
"diseased". The second set of subjects is simply those who do not
fall within the first set. Subjects in this second set may be
"non-diseased;" that is, normal subjects. Alternatively, subjects
in this second set may be selected to exhibit one symptom or a
constellation of symptoms that mimic those symptoms exhibited by
the "diseased" subjects. In still another alternative, this second
set may represent those at a different time point from disease
incidence. Preferably, data for the same set of markers is
available for each patient. This set of markers may include all
candidate markers which may be suspected as being relevant to the
detection of a particular disease or condition. Actual known
relevance is not required. Embodiments of the compositions, methods
and systems described herein may be used to determine which of the
candidate markers are most relevant to the diagnosis of the disease
or condition. The levels of each marker in the two sets of subjects
may be distributed across a broad range, e.g., as a Gaussian
distribution. However, no distribution fit is required.
[0235] 1. Acute Myocardial Infarct
[0236] The methods of the invention are especially useful in
diagnosis, prognosis, and/or treatment selection in patients
suspected of acute myocardial infarct (AMI). Single or serial
cardiac troponin measurements in patients suspected of AMI provide
incremental prognostic information that improves the prognosis and
indicates appropriate and early therapeutic intervention to
minimize the risk of adverse outcomes.
[0237] Thus, the invention provides a method of diagnosing,
predicting, and/or preventing or treating AMI in an individual by
assaying a sample from the individual, e.g., a blood sample, plasma
sample, and/or serum sample, for cardiac troponin, e.g., cTnI, and
detecting a concentration of cardiac troponin in the sample at a
limit of detection of less than about 50, 40, 30, 20, 15, 10, 9, 8,
7, 6, 5, 4, 3, 2, or 1 pg/ml, e.g., less than about 20 pg/ml,
wherein the concentration of cardiac troponin in the sample
indicates or predicts AMI. The cardiac troponin may be cTnI or
cTnT, and may be total troponin or a measure of a particular form,
e.g., free, complexed, or fragment; in some embodiments, a ratio of
one or more forms of the troponin is used, as described herein. In
some embodiments, total cTnI is measured in the sample or series of
samples. In some embodiments, total cTnT is measured in the sample
or series of samples. In some embodiments, total cTnI+cTnT is
measured in the sample or series of samples. In some embodiments,
the cardiac troponin level is determined at or near the time the
individual presents to a health professional with symptoms
indicative of AMI. Such symptoms include, but are not limited to,
chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme
levels, and shortness of breath.
[0238] In some embodiments, a series of measurements is taken, and
a spike in the cardiac troponin concentration in the samples
indicates, predicts, or provides a basis for prognosis of AMI. In
some embodiments, a spike of over 50%, over 100%, over 150%, over
200%, over 250%, over 300%, over 400%, or over 500% of baseline
indicates, predicts, or provides a basis for prognosis of AMI. In
some embodiments, a cardiac troponin level of over about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 pg/ml in a
single sample indicates, predicts, or provides a basis for
prognosis of AMI, regardless of baseline levels, if obtained. In
some embodiments, a cardiac troponin level of about 1-10, or about
5-15, or about 10-50, about 10-200, about 10-100, or about 10-40,
or about 15-50, or about 15-40, or about 20-200, about 20-150,
about 20-100, about 20-50, or about 20-40, or about 20-30 pg/ml
indicates, predicts, or provides a basis for prognosis of AMI.
[0239] In some embodiments diagnosis or prognosis includes
stratification for the individual, based on cardiac troponin
concentration in the sample or series of samples. Such
stratification may be based on the concentration of cardiac
troponin in single samples, presence of spikes and/or size of
spikes from baseline in a series of samples, ratios of different
forms of the cardiac troponin, absolute values for different forms
of cardiac troponin, rate of change in concentration for the
cardiac troponin or for one or more forms of the cardiac troponin
in a series of samples, change in ratios of different forms of
cardiac troponin over time in a series of samples, and any other
information based at least in part on cardiac troponin
concentration in the sample or series of samples. Stratification
may be based on values obtained from populations of normal and
diseased subjects, as described herein. Appropriate treatment may
also be determined based on the stratification of the
individual.
[0240] In some embodiments, concentration of cardiac troponin is
determined in combination with one or more other markers, e.g.,
markers of myocardial ischemia, myocardial infarct or markers of
stroke, and the concentrations of each marker are considered in
determining the diagnosis, prognosis, or method of treatment. Other
clinical indications typically will also be taken into account,
e.g., EKG, symptoms, history, and the like, as will be apparent to
those of skill in the art. Appropriate algorithms for diagnosis,
prognosis, or treatment may be constructed based on the
combinations of such markers and clinical indications in
combination with troponin levels.
[0241] Markers useful in combination with cardiac troponin in the
methods of the invention include but are not limited to creatine
kinase (CK) and its myocardial fraction CK myocardial band (MB),
aspartate aminotransferase, lactate dehydrogenase (LDH),
.alpha.-hydroxybutyrate dehaydrogenase, myoglobin, glutamate
oxaloacetate transaminase, glycogen phosphorylase BB, unbound free
fatty acids, heart fatty acid binding protein (H-FABP),
ischemia-modified albumin, myosin light chain 1, myosin light chain
2. Markers of inflammation and plaque instability useful in
combination with cardiac troponin in the methods of the invention
include but are not limited to C-reactive protein, white blood cell
count, soluble CD40 ligand, myeloperoxidase, monocyte
chemoattractant protein-1, whole blood choline, and
pregnancy-associated plasma protein A. Other markers of
inflammation may be detected, and include combinations of Il-8,
IL-1.beta., IL6, IL10, TNF, and IL-12p70, as well as other
cytokines or markers that will be apparent to those of skill in the
art.
[0242] In some embodiments, cardiac troponin, e.g., cTnI, is
measured together, e.g., in the same sample, or in samples from the
same individual taken at or near the same time, with a marker
selected from the group consisting of creatine kinase (CK) and its
myocardial fraction CK myocardial band (MB), aspartate
aminotransferase, lactate dehydrogenase (LDH),
.alpha.-hydroxybutyrate dehaydrogenase, myoglobin, glutamate
oxaloacetate transaminase, glycogen phosphorylase BB, unbound free
fatty acids, heart fatty acid binding protein (H-FABP),
ischemia-modified albumin, myosin light chain 1, and myosin light
chain 2. In some embodiments cardiac troponin, e.g., cTnI, is
measured together with CK-MB, e.g., in the same sample, or in
samples from the same individual taken at or near the same
time.
[0243] In some embodiments, cardiac troponin, alone or in
combination with other markers or clinical signs, measured as
described herein, is used to determine reinfarction. In some
embodiments, cardiac troponin, alone or in combination with other
markers or clinical signs, measured as described herein, is used to
determine characteristics of an infarct, e.g., size, or duration
since infarct. In the latter case, fragments of troponin produced
by proteolysis in the blood may be compared to total troponin; the
greater the proportion of fragments, the more time has elapsed
since infarct.
[0244] 2. Conditions Other than AMI
[0245] The methods of the invention also include methods of
diagnosis, prognosis, and treatment based on concentration of
cardiac troponin in a sample that are useful in conditions other
than AMI.
[0246] Many conditions include potential or actual cardiac damage,
and the ability to measure cardiac troponin at the levels described
herein allow for early detection of such damage and early
intervention. Knowledge of the concentration of cardiac troponin as
measured by the methods and compositions of the invention is useful
in diagnosis, prognosis, and determination of treatment for such
conditions. Conditions include percutaneous coronary interventions,
cardiac surgery, heart failure, acute rheumatic fever, amyloidosis,
cardiac trauma (including contusion, ablation, pacing, firing,
cardioversion, catheterization and cardiac surgery), reperfusion
injury, cardiotoxicity from cancer therapy, congestive heart
failure, end-stage renal failure, glycogen storage disease type II
(Pompe's disease), heart transplantation, haeomoglobinopathy with
transfusion haemosiderosis, hypertension, including gestational
hypertension, hypotension, often with arrhythmias, hypothyroidism,
myocarditis, pericarditis, post-operative non-cardiac surgery,
pulmonary embolism, and sepsis.
[0247] In these embodiments, the troponin levels may be determined
concomitantly with the levels of marker(s) that are specific for
the non-cardiac disease, or other symptoms or clinical signs of the
disease; the marker(s) concentration and/or information regarding
other symptoms or clinical signs is combined with information
regarding cardiac troponin concentrations, determined as described
herein, to determine a diagnosis, prognosis, and/or method of
treatment. For example, embodiments of the invention may employ, in
addition to determination of cardiac troponin concentration,
determination of the concentration of one or more of the
polypeptides referenced above, or other protein markers useful in
diagnosis, prognosis, or differentiation of disease. In some
embodiments, a panel of markers for the disease is provided, where
the panel includes cardiac troponin concentration, as described
herein, and at least on other marker for the disease. The panel may
include, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual
markers, which including one or more cardiac troponins, e.g., total
cTnI. The analysis of a single marker or subsets of marker can be
carried out by one skilled in the art to optimize clinical
sensitivity or specificity in various clinical settings. These
include, but are not limited to ambulatory, urgent care, critical
care, intensive care, monitoring unit, inpatient, outpatient,
physician office, medical clinic, and health screening settings.
Furthermore, one skilled in the art can use a single marker or a
subset of markers in combination with an adjustment of the
diagnostic threshold in each of the aforementioned settings to
optimize clinical sensitivity and specificity.
[0248] a. Cardiac toxicity The compositions and methods of the
invention are especially useful in determining and monitoring
cardiac toxicity that results from a treatment, e.g., cardiac
toxicity of drug treatment. Thus, for example, the invention
provides a method of assessing cardiac toxicity of a treatment by
measuring cardiac troponin in an individual by i) determining a
concentration of cardiac troponin in a sample or determining the
concentrations of cardiac troponin in a series of samples from the
individual, where at least one of the samples is taken from the
individual during or after a time when the individual is receiving
the treatment, where the concentration or concentrations is
determined by a cardiac troponin assay with a limit of detection
for the cardiac troponin in said sample of less than about 50, 40,
30, 10, 5, 4, 3, 2 or 1 pg/ml, e.g., less than about 20 pg/ml; and
ii) assessing the degree of cardiac toxicity of the treatment based
on said concentration or concentrations. In some embodiments, the
treatment is a drug treatment. In some embodiments, the treatment
is a non-drug treatment. The method of determining the
concentration of cardiac troponin includes any suitable method with
the requisite sensitivity, e.g., the methods described herein. In
some embodiments, the methods utilize a method of determining a
concentration of cardiac troponin in the sample where the method
comprises detecting single molecules of troponin, or complexes or
fragments thereof.
[0249] Especially useful are methods of determination of cardiac
toxicity utilizing the cross-reacting antibodies described herein,
i.e., antibodies that react with troponin from at least two
species, such as humans and another species such as rat, dog,
mouse, or monkey. Such antibodies may be used in animal studies of
drug toxicity, where the individual for which toxicity is assessed
is, e.g., a mammal, such as a rat, mouse, dog, monkey, or other
animal used in such studies. Toxicity in various species may be
directly compared when the antibody used in the assay is the same
antibody, thus reducing variability.
[0250] It will be appreciated that the compositions and methods of
the invention may be used in conjunction with specific drugs whose
side effects include cardiotoxicity in order to monitor the cardiac
toxicity. Thus, the invention provides methods of monitoring
cardiac toxicity in an individual who is receiving a drug that is
known to cause cardiac toxicity by determining the concentration of
cardiac troponin in one or more samples obtained from the
individual, where the concentration or concentrations is determined
by a cardiac troponin assay with a limit of detection for the
cardiac troponin in said sample or samples of less than about 50,
40, 30, 10, 5, 4, 3, 2 or 1 pg/ml, e.g., less than about 20 pg/ml;
and ii) assessing the degree of cardiac toxicity of the drug
treatment based on said concentration or concentrations. In some
embodiments the method further includes a step iii) determining
whether or not to continue the drug treatment based on the
assessment of step ii). Drugs whose side effects include cardiac
toxicity are well-known in the art.
[0251] C. Serial Testing
[0252] A clinical need for very high sensitivity troponin is
developing and with it the development of highly sensitive assays
for the detection of troponin as described herein. There are three
major areas where next-generation troponin assays, e.g. assays for
cTnI, can potentially improve current practice. These ways include
the earlier diagnosis of an individual than is currently possible
with existing routine markers e.g., with troponin, Creatine
kinase-MB, and myoglobin, improved risk stratification for future
adverse cardiac events among AMI ruleouts, and monitoring of
therapeutic drugs that have the potential to cause cardiotoxicity.
For AMI patients, the first detectable increase in troponin after
the onset of chest pain will enable an earlier diagnosis and triage
of patients to the appropriate level of cardiac care. The
sensitivity of the analyzer, compositions, and assays described
herein has the improved analytical sensitivity and precision to
lower the 99th percentile cutoff value. In particular the analyzer
system can be used to detect low concentrations of troponin levels
and to detect the biological variability (the variability between
samples), reference change values, and the index of individuality
between samples. The reference change value (RCV) is used to
interpret serial results. For serial results to be significantly
different, the difference in the numerical results must be greater
than the combined variation inherent in the two results. The index
of individuality corresponds to the ratio of the within subject
biological variation to the between-subject variation. Increasing
the analytical sensitivity will result in a lowering of the cutoff
concentration at the 99th percentile recommended by The Joint
European Society of Cardiology/American College of Cardiology
committee for the Redefinition of Myocardial Infarction, and will
result in a higher frequency of positive troponin results in
patients who present to the emergency room with chest pain. Unless
ischemia can be ruled out by clinical or other means, a mildly
increased detected level of troponin will produce more false
positive cases of AMI. In order to reduce the diagnostic confusion
caused by high sensitivity troponin assays, the National Academy of
Clinical Biochemistry advocate the use of serial testing, using a
more rigorous determination of statistically significant reference
change values that are based on the detection or measurement of an
analyte's biologic variation in a patient.
[0253] The determination of the biological variation for cardiac
troponin has not been previously possible due to the lack of
analytical sensitivity of assays to reliably measure troponin in
the blood of healthy subjects. The availability of high sensitivity
troponin assays now enables the reliable detection of troponin due
to the normal turnover of cardiac myocytes with the requisite
imprecision (.ltoreq.10%). The biological variation data does not
influence the interpretation of patients who have a very high
troponin concentration, e.g., a 10-fold increases relative to the
99th percentile cutoff. These patients should be immediately
admitted and treated without a need for serial troponin testing. In
contrast, proper use of the biological variation will be important
in the interpretation of minor increases of troponin, i.e., values
at or just above the 99th percentile limit when very high
sensitivity assays are used. Serial cardiac troponin values that
exceed the upper RCV in the clinical context of chest pain increase
the likelihood that the patient is suffering an evolving AMI. This
interpretation may be valid even if all serial troponin results are
below the population-based reference limit (due to the low index of
individuality). However, there may be other acute disease processes
that can increase troponin concentrations over the short term, such
as sepsis. Patients with serial troponin values that are mildly
increased but are unchanging would increase the likelihood that a
chronic cardiac condition is present that is known to cause cardiac
damage (e.g., myocarditis, heart and kidney failure). Patients with
declining serial troponin results might indicate a resolving AMI,
particularly if there is a history of chest pain from the prior
days or week. It should be noted that patients with a positive
troponin due to a chronic disease can present with an acute
exacerbation and increasing troponin values that mimic an AMI
release pattern. Therefore clinical judgment remains paramount in
the interpretation of troponin testing.
[0254] Using the highly sensitive analyzers described previously,
an individual having one or more symptoms of cardiac infarct can be
classified with a series of steps comprising: i) obtaining a sample
from said individual, ii) detecting the level of troponin in the
sample and iii) if the level of troponin is above 10-fold of the
99th percentile, taking a first action with respect to the
individual and if the level of troponin is below 10-fold of the
99th percentile taking a second action. In some embodiments, the
first action is taken if patient has about a 3-fold to about a
10-fold increase relative to the 99th percentile cutoff. In some
embodiments, the first action is taken if patient has about a
4-fold to about a 5-fold increase relative to the 99th percentile
cutoff. In some embodiments, the first action is taken if patient
has about a 6-fold to about a 7-fold increase relative to the 99th
percentile cutoff. In some embodiments, the first action is taken
if patient has about an 8-fold to about a 10-fold increase relative
to the 99th percentile cutoff. In some embodiments, the first
action is taken if patient has about a 1.5-fold to about a 2-fold
increase. In some embodiments, the first action is taken if patient
has about a 2-fold to about a 2.5-fold increase. In some
embodiments, the first action is taken if patient has about a
2.5-fold to about a 3-fold increase. In some embodiments, the first
action is taken if patient has about a 3-fold to about a 3.5-fold
increase. In some embodiments, the first action is taken if patient
has about a 3.5-fold to about a 4-fold increase. In some
embodiments, the first action is taken if patient has about a
4-fold to about a 4.5-fold increase. In some embodiments, the first
action is taken if patient has about a 4.5-fold to about a 5-fold
increase. In some embodiments, the first action is taken if patient
has about a 5-fold to about a 5.5-fold increase. In some
embodiments, the first action is taken if patient has about a
5.5-fold to about a 6-fold increase. In some embodiments, the first
action is taken if patient has about a 6-fold to about a 6.5-fold
increase. In some embodiments, the first action is taken if patient
has about a 6.5-fold to about a 7-fold increase. In some
embodiments, the first action is taken if patient has about a
7-fold to about a 7.5-fold increase. In some embodiments, the first
action is taken if patient has about a 7.5-fold to about an 8-fold
increase. In some embodiments, the first action is taken if patient
has about a 8-fold to about a 8.5-fold increase. In some
embodiments, the first action is taken if patient has about an
8.5-fold to about a 9-fold increase. In some embodiments, the first
action is taken if patient has about a 9-fold to about a 9.5-fold
increase. In some embodiments, the first action is taken if patient
has about a 9.5-fold to about a 10-fold increase. In some
embodiments the first action is taken if the increase is more than
about 2-fold. In some embodiments the first action is taken if the
increase is more than about 3-fold. In some embodiments the first
action is taken if the increase is more than about 4-fold. In some
embodiments the first action is taken if the increase is more than
about 5-fold. In some embodiments the first action is taken if the
increase is more than about 6-fold. In some embodiments the first
action is taken if the increase is more than about 7-fold. In some
embodiments the first action is taken if the increase is more than
about 8-fold. In some embodiments the first action is taken if the
increase is more than about 9-fold. In some embodiments the first
action is taken if the increase is more than about 10-fold.
[0255] In some embodiments, the individual is a patient being
evaluated for a possible cardiac event. In such a case, the first
action can be admission of the individual to a hospital, or other
appropriate clinical action for treatment of the cardiac event. The
second action can be to hold the individual for a period of time
for further observation. During the time the individual is held a
series of samples are taken from said patient and the level of
troponin in each sample detected. In some embodiments, the interval
between samples is less than about 4, 3, 2, 1, 0.5 hours apart. In
some embodiments, the sample interval is less than about 4 hours
apart. In some embodiments, the sample interval is about 1 hour. In
some embodiments, the sample interval is about 2 hours. It will be
recognized that circumstances in the clinic dictate sample
intervals and some degree of variation is acceptable and is
encompassed within the invention, as long as results may be
reliably interpreted. In some embodiments, in addition to the level
of troponin detected in each sample, the rate of change in the
level of troponin over two or more samples in the series of samples
is detected. In some embodiments, the change in the level of
troponin is a decrease. In some embodiments, the change in the
level of troponin is an increase. A decision can be made regarding
a course of action for said individual based on said rate of
change, e.g., if the rate of change in troponin values exceeds a
predetermined upper reference rate of change value, the decision is
to admit the individual. It will be recognized that other clinical
factors well-known to those of skill in the art can be used to
modify the decision based on troponin levels, e.g., if other
clinical manifestations of acute myocardial infarct are present,
such as abnormal EKG, abnormal enzyme levels, physical symptoms,
and the like, then the threshold for troponin that leads to an
admission is suitably decreased.
[0256] The decision to take a first action, e.g., to admit a
patient, can also be made if one or more spikes in the level of
troponin are seen from sample to sample. The spike can be any
increase in the level of troponin detected compared to one or more
samples wherein the increase is compared to a threshold level. For
example, if the threshold level is determined to be 10 pg/ml and if
in one of the sample of the series of samples the troponin level
detected is 15 pg/ml, the patient is admitted. In some embodiments,
the threshold level is determined from a reference population. In
some embodiments, the threshold level is set at a level for an
individual, e.g., the initial level of troponin detected from the
first sample taken from the patient. In these embodiments, a
relative increase indicates a spike, no matter what the absolute
values are. In some embodiments, a spike is determined when the
troponin level increases 2, 3, 4, 5, 6, 7, 8, 9 10, or more than
10-fold above a reference value for the individual, e.g., a value
set at the troponin level in the first sample taken from the
individual. Alternatively, a spike in the level of troponin can be
seen when comparing troponin levels between samples from the
individual. In such a case, no matter what the value of the level
of troponin detected, the patient is admitted if the value
increases by more than a certain amount, e.g., a patient is
admitted when the level of troponin increases by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, or more than 10 pg/ml as compared to one or more
neighboring samples. In some embodiments, the change in the level
of troponin indicates an acute cardiovascular disease. In some
embodiments, the change in the level of troponin indicates a
chronic cardiovascular disease. In some embodiments, the change in
the level of troponin indicates cardiotoxicity.
[0257] The existing commercial assays lack the sensitivity to low
to detect very minor and chronic increases. With the development of
high sensitive assays, the utility of cardiac troponin should be
re-examined for this application. If proven to be useful, the
long-term biological variation and calculation of the RCV will be
relevant.
[0258] D. Business Methods
[0259] The present invention relates to systems and methods
(including business methods) for establishing markers of cardiac
troponin that can be used for diagnosing, prognosing, or
determining a method of treatment of a biological state or a
condition in an organism, preparing diagnostics based on such
markers, and commercializing/marketing diagnostics and services
utilizing such diagnostics. The biological state may be acute
myocardial infarct, or cardiac damage due to drug toxicity, or
non-AMI states as described herein.
[0260] In one embodiment, the business methods herein comprise:
establishing one or more cardiac troponin markers using a method
comprising: establishing a range of concentrations for said marker
or markers in biological samples obtained from a first population
by measuring the concentrations of the marker or markers in the
biological samples by detecting single molecules of the marker or
markers at a level of detection of less than about 50, 20, 10, 5,
or 1 pg/ml; and commercializing the one or more markers established
in the above step, e.g., in a diagnostic product. The diagnostic
product herein can include one or more antibodies that specifically
binds to the cardiac troponin marker and a fluorescent moiety that
is capable of emitting an average of at least about 200 photons
when simulated by a laser emitting light at the excitation
wavelength of the moiety, where the laser is focused on a spot of
not less than about 5 microns in diameter that contains the moiety,
and wherein the total energy directed at the spot by the laser is
no more than about 3 microJoules.
[0261] In one embodiment, the business methods herein comprise:
establishing a range of normal values for a cardiac troponin marker
using a system comprising: establishing a range of concentrations
for said cardiac troponin marker in biological samples obtained
from a first population by measuring the concentrations of the
marker the biological samples by detecting single molecules of the
marker at a level of detection less than about 50, 20, 10, 5, or 1
pg/ml; and providing a diagnostic service to determine if an
organism has or does not have a state or condition of interest,
e.g., AMI, cardiac toxicity due to drug treatment, or a non-AMI
condition. A diagnostic service herein may be provided by a CLIA
approved laboratory that is licensed under the business or the
business itself. The diagnostic services herein can be provided
directly to a health care provider, a health care insurer, or a
patient. Thus the business methods herein can make revenue from
selling e.g., diagnostic services or diagnostic products.
[0262] The business methods herein also contemplate providing
diagnostic services to, for example, health care providers,
insurers, patients, etc. The business herein can provide diagnostic
services by either contracting out with a service lab or setting up
a service lab (under Clinical Laboratory Improvement Amendment
(CLIA) or other regulatory approval). Such service lab can then
carry out the methods disclosed herein to identify if a cardiac
troponin marker is within a sample.
VII. Compositions
[0263] The invention provides compositions useful in the detection
and quantitation of cardiac troponin. Compositions include binding
partners to cardiac troponin that are labeled with suitable labels
for detection by the methods of the invention, pairs of binding
partners in which one or both of the binding partners are labeled
with suitable labels for detection by the methods of the invention,
solid supports to which capture binding partners are attached, in
some embodiments also with detection binding partners.
[0264] Exemplary embodiments include a composition for the
detection of cardiac troponin that includes a binding partner to
the cardiac troponin attached to a fluorescent moiety, where the
fluorescent moiety is capable of emitting an average of at least
about 200 photons when simulated by a laser emitting light at the
excitation wavelength of the moiety, where the laser is focused on
a spot of not less than about 5 microns in diameter that contains
the moiety, and wherein the total energy directed at the spot by
the laser is no more than about 3 microJoules. In some embodiments,
the binding partner includes an antibody to the cardiac troponin.
In some embodiments, the antibody is a polyclonal antibody. In some
embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is a cross-reacting antibody, e.g., an
antibody that cross-reacts with cardiac troponin from at least two
species, e.g., at least two species selected from the group
consisting of human, monkey, dog, and mouse. In some embodiments
the antibody cross-reacts with cardiac troponins from all of human,
monkey, dog, and mouse. In some embodiments, the cardiac troponin
is selected from the group consisting of cTnI and cTnT. In some
embodiments, the cardiac troponin is cTnI. In some embodiments,
cardiac troponin is cTnT. The antibody may specific to a specific
region of the troponin molecule, e.g., specific to a region
comprising amino acids 27-41 of cardiac troponin I. The fluorescent
moiety may contain one or more molecules that comprises at least
one substituted indolium ring system in which the substituent on
the 3-carbon of the indolium ring contains a chemically reactive
group or a conjugated group. The label composition may include a
fluorescent moiety that includes one or more dye molecules selected
from the group consisting of AlexaFluor 488, 532, 647, 700, or 750.
The label composition may include a fluorescent moiety that
includes one or more dye molecules selected from the group
consisting of AlexaFluor 488, 532, 700, or 750. The label
composition may include a fluorescent moiety that includes one or
more dye molecules that are AlexaFluor 488. The label composition
may include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 555. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 610. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 647. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 680. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 700. The label composition may
include a fluorescent moiety that includes one or more dye
molecules that are AlexaFluor 750.e
[0265] In some embodiments, the invention provides a composition
that includes a set of standards for the determination of a
concentration of a cardiac troponin, wherein at least one of the
standards is at a concentration of cardiac troponin less than about
20, 15, 10, 5, 4, 3, 2, or 1 pg/ml. In some embodiments, the
invention provides a composition that includes a set of standards
for the determination of a concentration of a cardiac troponin,
wherein at least one of the standards is at a concentration of
cardiac troponin less than about 20 pg/ml. In some embodiments, the
invention provides a composition that includes a set of standards
for the determination of a concentration of a cardiac troponin,
wherein at least one of the standards is at a concentration of
cardiac troponin less than about 10 pg/ml. In some embodiments, the
invention provides a composition that includes a set of standards
for the determination of a concentration of a cardiac troponin,
wherein at least one of the standards is at a concentration of
cardiac troponin less than about 5 pg/ml. In some embodiments, the
invention provides a composition that includes a set of standards
for the determination of a concentration of a cardiac troponin,
wherein at least one of the standards is at a concentration of
cardiac troponin less than about 1 pg/ml.
[0266] Other compositions of the invention are as described herein.
VIII. Kits
[0267] The invention further provides kits. Kits of the invention
include one or more compositions useful for the sensitive detection
of cardiac troponin, as described herein, in suitable packaging. In
some embodiments kits of the invention provide labels, e.g.,
binding partner such as an antibody that is specific for cardiac
troponin, where the binding partner is attached to a fluorescent
moiety. In some embodiments kits of the invention provide binding
partner pairs, e.g., antibody pairs, that are specific for cardiac
troponin, where at least one of the binding partners is a label for
a cardiac troponin, as described herein. In some embodiments, the
binding partners, e.g., antibodies, are provided in separate
containers. In some embodiments, the binding partners, e.g.,
antibodies, are provided in the same container. In some
embodiments, one of the binding partners, e.g., antibody, is
immobilized on a solid support, e.g., a microtiter plate or a
paramagnetic bead. In some of these embodiments, the other binding
partner, e.g., antibody, is labeled with a fluorescent moiety as
described herein.
[0268] Binding partners, e.g., antibodies, solid supports, and
fluorescent labels for components of the kits may be any suitable
such components as described herein.
[0269] The kits may additionally include reagents useful in the
methods of the invention, e.g., buffers and other reagents used in
binding reactions, washes, buffers or other reagents for
preconditioning the instrument on which assays will be run, and
elution buffers or other reagents for running samples through the
instrument.
[0270] Kits may include one or more standards, e.g., standards for
use in the assays of the invention, such as standards of highly
purified, e.g., recombinant, human cTnI or human cTnT, or various
fragments, complexes, and the like, thereof. Kits may further
include instructions.
EXAMPLES
[0271] The following examples are offered by way of illustration
and not by way of limiting the remaining disclosure.
[0272] Unless otherwise specified, processing samples in the
Examples were analyzed in a single molecule detector (SMD) as
described herein, with the following parameters: Laser: continuous
wave gallium arsenite diode laser of wavelength 639 nm (Blue Sky
Research, Milpitas, Calif.), focused to a spot size of
approximately 2 microns (interrogation space of 0.004 pL as defined
herein); flow rate=5 microliter/min through a fused silica
capillary of 100 micron square ID and 300 micron square OD;
non-confocal arrangement of lenses (see, e.g., FIG. 1A); focusing
lens of 0.8 numerical aperture (Olympus); silicon avalanche
photodiode detector (Perkin Elmer, Waltham, Mass.).
Example 1
Sandwich Assays for Biomarkers: Cardiac Troponin I (cTnI)
[0273] The assay: The purpose of this assay was to detect the
presence of cardiac Troponin I (cTnI) in human serum. The assay
format was a two-step sandwich immunoassay based on a mouse
monoclonal capture antibody and a goat polyclonal detection
antibody. Ten microliters of sample were required. The working
range of the assay is 0-900 pg/ml with a typical analytical limit
of detection of 1-3 pg/ml. The assay required about four hours of
bench time to complete.
[0274] Materials: the following materials were used in the
procedure described below: Assay plate: Nunc Maxisorp, product
464718, 384 well, clear, passively coated with monoclonal antibody,
BiosPacific A34440228P Lot # A0316 (5 .mu.g/ml in 0.05 M sodium
carbonate pH 9.6, overnight at room temperature); blocked with 5%
sucrose, 1% BSA in PBS, and stored at 4 oC. For the standard curve,
Human cardiac Troponin I (BiosPacific Cat # J34000352) was used.
The diluent for the standard concentrations was human serum that
was immonodepleted of endogenous cTnI, aliquoted and stored at -20
oC. Dilution of the standards was done in a 96 well, conical,
polypropylene, (Nunc product #249944). The following buffers and
solutions were used: (a) assay buffer: BBS with 1% BSA and 0.1%
TritonX-100; (b) passive blocking solution in assay buffer
containing 2 mg/ml mouse IgG, (Equitech Bio); 2 mg/ml goat IgG,
(Equitech Bio); and 2 mg/ml MAK33 poly, Roche #11939661; (c)
detection Antibody (Ab): Goat Polyclonal antibody affinity purified
to Peptide 3, (BiosPacific G129C), which was label with a
fluorescent dye AlexaFluor 647, and stored at 4 oC.; detection
antibody diluent: 50% assay buffer, 50% passive blocking solution;
wash buffer: borate buffer saline Triton Buffer (BBST) (1.0 M
borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); elution
buffer: BBS with 4M urea, 0.02% Triton X-100 and 0.001% BSA.
[0275] Preparation of AlexaFluor 647 labeled antibodies: the
detection antibody G-129-C was conjugated to AlexaFluor 647 by
first dissolving 100 ug of G-129-C in 400 uL of the coupling buffer
(0.1M NaHCO.sub.3). The antibody solution was then concentrated to
50 ul by transferring the solution into YM-30 filter and subjecting
the solution and filter to centrifugation. The YM-30 filter and
antibody was then washed three times by adding 400 ul of the
coupling buffer. The antibody was recovered by adding 50
.quadrature.l to the filter, inverting the filter, and centrifuging
for 1 minute at 5,000.times.g. The resulting antibody solution was
1-2 ug/ul. AlexaFluor 647 NHS ester was reconstituted by adding 20
ul DMSO to one vial of AlexaFluor 647, this solution was stored at
-20 oC. for up to one month. 3 ul of AlexaFluor 647 stock solution
was added to the antibody solution, which was then mixed and
incubated in the dark for one hour. After the one hour, 7.5 ul 1M
tris was added to the antibody AlexaFluor 647 solution and mixed.
The solution was ultramiltered with YM-30 to remove low molecular
weight components. The volume of the retentate, which contained the
antibody conjugated to AlexaFluor 647, was adjusted to 200-400
.quadrature.l by adding PBS. 3 ul 10% NaN3 was added to the
solution, the resulting solution was transferred to an Ultrafree
0.22 centrifugal unit and spun for 2 minutes at 12,000.times.g. The
filtrate containing the conjugated antibody was collected and used
in the assays.
[0276] Procedure: cTnI standard and sample preparation and
analysis:
[0277] The standard curve was prepared as follows: working
standards were prepared (0-900 pg/ml) by serial dilutions of the
stock of cTnI into standard diluent or to achieve a range of cTnI
concentrations of between 1.2 pg/ml 4.3 .mu.g/ml.
[0278] 10 .mu.l passive blocking solution and 10 .mu.l of standard
or of sample were added to each well. Standards were run in
quadruplicate. The plate was sealed with Axyseal sealing film,
centrifuged for 1 min at 3000 RPM, and incubated for 2 hours at
25.degree. C. with shaking. The plate was washed five times, and
centrifuged until rotor reached 3000 RPM in an inverted position
over a paper towel. A 1 nM working dilution of detection antibody
was prepared, and 20 .mu.l detection antibody were added to each
well. The plate was sealed and centrifuged, and the assay incubated
for 1 hour at 25.degree. C. with shaking. 30 .mu.l elution buffer
were added per well, the plate was sealed and the assay incubated
for 1/2 hour at 25.degree. C. The plate was either stored for up to
48 hours at 4.degree. C. prior to analysis, or the sample was
analyzed immediately.
[0279] For analysis, 20 .mu.l per well were acquired at 40
.mu.l/minute, and 5 .mu.l were analyzed at 5 .mu.l/minute. The data
were analyzed based on a threshold of 4 sigma. Raw signal versus
concentration of the standards was plotted. A linear fit was
performed for the low concentration range, and a non-linear fit was
performed for the full standard curve. The limit of detection (LoD)
was calculated as LOD=(3.times. standard deviation of zeros)/slope
of linear fit. The concentrations of the samples were determined
from the equation (linear or non-linear) appropriate for the sample
signal.
[0280] An aliquot was pumped into the analyzer.
Individually-labeled antibodies were measured during capillary flow
by setting the interrogation volume such that the emission of only
1 fluorescent label was detected in a defined space following laser
excitation. With each signal representing a digital event, this
configuration enables extremely high analytical sensitivities.
Total fluorescent signal is determined as a sum of the individual
digital events. Each molecule counted is a positive data point with
hundreds to thousands of DMC events/sample. The limit of detection
the cTnI assay of the invention was determined by the mean +3 SD
method.
[0281] Results: Data for a typical cTnI standard curve measured in
quadruplicate using the assay protocol is shown in Table 2.
TABLE-US-00003 TABLE 2 Standard Curve for cTnI cTnI Standard
(pg/ml) Signal Deviation % CV 0 233 25 10.8 1.5625 346 31 8.9 3.125
463 35 7.5 6.25 695 39 5.6 12.5 1137 61 5.3 25 1988 139 7.0 50 3654
174 4.8 100 5493 350 6.4 200 8264 267 3.2 400 9702 149 1.5 800 9976
50 0.5
[0282] The sensitivity of the analyzer system was tested in 15 runs
and was found routinely to detect sub femtomol/l (fM) levels of
calibrator, as shown by the data in Table 3. The precision was 10%
at 4 and 12 pg/ml cTnI.
TABLE-US-00004 TABLE 3 Instrument Sensitivity Calibrator Signal
(fM) counts CV 0 11 12 302 9 60 1341 8 300 4784 7
[0283] Linearized standard curve for the range concentrations of
cTnI are shown in FIG. 5.
[0284] The analytical limit of detection (LoD) was determined
across 15 sequential assays. The LoD was the mean of the 0 std+3 SD
(n=4) intra-assay determinations. The average LoD was 1.7 pg/ml
(range 0.4-2.8 pg/ml).
[0285] The recovery of the sample was determined by analyzing
samples of serum that had been immunodepleted of cTnI and spiked
with known amounts of cTnI. Table 4 shows the data for sample
recovery by the system analyzed over 3 days.
TABLE-US-00005 TABLE 4 Sample Recovery Spike Recovery Standard %
(pg/ml) (mean) Deviation CV 5 5.7 0.9 16 15 13.7 0.2 2 45 43 0.6 2
135 151 6.2 4
[0286] The linearity of the assay was determined in pooled human
serum that was spiked with cTnI and diluted with standard diluent.
The results in Table 5 show the dilutions and % of the signal
expected for the corresponding dilution.
TABLE-US-00006 TABLE 5 Assay Linearity Serum % of Dilution expected
1:2 79 1:4 87 1:8 96
[0287] These data show that the analyzer system of the invention
allows for performing highly sensitive laser-induced immunoassay
for sub-femtomolar concentrations of cTnI.
Example 2
Sandwich Bead-Based Assays for TnI
[0288] The assays described above use the same microtiter plate
format where the plastic surface is used to immobilize target
molecules. The single particle analyzer system also is compatible
with assays done in solution using microparticles or beads to
achieve separation of bound from unbound entities.
[0289] Materials: MyOne Streptavidin C1 microparticles (MPs) are
obtained from Dynal (650.01-03, 10 mg/ml stock). Buffers use in the
assay include: 10.times. borate buffer saline Triton Buffer (BBST)
(1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3);
assay buffer (2 mg/ml normal goat IgG, 2 mg/ml normal mouse IgG,
and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 M Tris (pH 8.1), 0.025 M
EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.1% NaN3,
stored at 4 C); sand elution buffer (BBS with 4 M urea, 0.02%
Triton X-100, and 0.001% BSA, stored at 2-8 C). Antibodies used in
the sandwich bead-based assay include: Bio-Ab (A34650228P
(BiosPacific) with 1-2 biotins per IgG) and Det-Ab (G-1129-C
(BiosPacific) conjugated to A647, 2-4 fluors per IgG). The standard
is recombinant human cardiac troponin I (BiosPacific, cat
#J34120352). The calibrator diluent is 30 mg/ml BSA in TBS
wEDTA.
[0290] Microparticles Coating: 100 ul of the MPs stock is placed in
an eppendorf tube. The MPs are washed three times with 100 ul of
BBST wash buffer by applying a magnet, removing the supernatant,
removing the magnet, and resuspending in wash buffer. After the
washes the MPs are resuspended in 100 ul of assay buffer and 15 ug
of Bio-Ab are added. The mixture is then incubated for an hour at
room temperature with constant mixing. The MPs are washed five
times with 1 ml wash buffer as described above. After the washes
the MPs are resuspended in 15 ml of assay buffer (or 100 ul to
store at 4 0C.).
[0291] Preparation of Standard and Samples: The standard is diluted
with calibrator diluent to prepare proper standard curve (usually
200 pg/ml down to 0.1 pg/ml). Frozen serum and plasma samples need
to be centrifuged 10 minutes at room temperature at 13 K rpm.
Clarified serum/plasma is removed carefully to avoid taking any
possible pellets or floaters and put into fresh tubes. 50 ul of
each standard or sample is pipetted into appropriate wells.
[0292] Capture Target: 150 ul of MPs (after resuspension to 15 ml
in assay buffer+400 mM NaCl) are added to each well. The mixture is
incubated on JitterBug, 5 at room temperature for 1 hr.
[0293] Washes and Detection: The plate is placed on a magnet and
the supernatant is removed after ensuring that all MPs are captured
by the magnet 250 ul of wash buffer are added after removing the
plate from the magnet. The plate is then placed on the magnet and
the supernatant is removed after ensuring that all MPs are captured
by the magnet. 20 ul Det-Ab are added per well (Det-Ab to 500 ng/ml
is diluted in assay buffer+400 mM NaCl)). The mixture is incubated
on JitterBug, 5 at room temperature for 30 min.
[0294] Washes and Elution: The plate is placed on a magnet and
washed three times with wash buffer. The supernatant is removed
after ensuring that all MPs are captured by the magnet and 250 ul
of wash buffer are added. After the washes the samples are
transferred into a new 96-well plate. The new plate is then placed
on the magnet and the supernatant is removed after ensuring that
all MPs are captured by the magnet. 250 ul of wash buffer are then
added after removing the plate from the magnet. The plate is then
placed on the magnet and the supernatant is removed after ensuring
that all MPs are captured by the magnet. 20 ul of elution buffer
are then added and the mixture is incubated on JitterBug, 5 at room
temperature for 30 min.
[0295] Filter out MPs and transfer to 384-well plate: The standard
and samples are transferred into a 384-well filter plate placed on
top of a 384-well assay plate. The plate is then centrifuged at
room temperature at 3000 rpm with a plate rotor. The filter plate
is removed and the appropriate calibrators are added. The plate is
covered and is ready to be run on SMD.
[0296] SMD: An aliquot is pumped into the analyzer.
Individually-labeled antibodies are measured during capillary flow
by setting the interrogation volume such that the emission of only
1 fluorescent molecule is detected in a defined space following
laser excitation. With each signal representing a digital event,
this configuration enables extremely high analytical sensitivities.
Total fluorescent signal is determined as a sum of the individual
digital events. Each molecule counted is a positive data point with
hundreds to thousands of DMC events/sample. The limit of detection
the cTnI assay of the invention is determined by the mean +3 SD
method.
Example 3
Concentration Range for cTnI in a Population of Normal Non-Diseased
Subjects
[0297] A reference range or normal range for cTnI concentrations in
human serum was established using serum samples from 88 apparently
healthy subjects (non-diseased). A sandwich immunoassay as
described in Example 1 was performed and the number of signals or
events as described above were counted using the single particle
analyzer system of the invention. The concentration of serum
troponin I was determined by correlating the signals detected by
the analyzer with the standard curve as described above. All assays
were perfumed in quadruplicate.
[0298] In accordance with recommendations by the current European
and American Cardiology Societies (ESC/ACC) troponin assays should
quantify accurately the 99th percentile of the normal range with an
assay imprecision (CV) of less than 10% in order to distinguish
reliably between patients with ACS and patients without ischemic
heart disease, and risk stratification for adverse cardiac events.
The assay showed that the biological threshold (cutoff
concentration) for TnI is at a TnI concentration of 7 pg/ml, which
is established at the 99th percentile with a corresponding CV of
10% (FIG. 5). At the 10% CV level the precision profile points at a
TnI concentration of 4 and 12 pg/ml.
[0299] In addition, the assay correlates well with the Troponin-I
standard measurements provided by the National Institute of
Standards and Technology (FIG. 6).
[0300] The assay of the invention is sufficiently sensitive and
precise to fulfill the requirements of the ESC/ACC, and it is the
most sensitive assay for cardiac troponin I when compared to assays
such as those described by Koerbin et al. (Ann Clin Biochem,
42:19-23 (2005). The assay of the invention has a 10-20 fold
greater sensitivity than that currently available assays, which has
determined the biological threshold range to be 111-333 pg/ml
cTnI.
Example 4
Detection of Early Release of TnI into the Circulation of Patients
with Acute Myocardial Infarction (AMI)
[0301] Study 1: 47 samples were obtained serially from 18 patients
that presented with chest pain in the emergency department (ED).
These patients all had non-ST elevated ECG were, and were diagnosed
with AMI. The concentration of cTnI in the initial samples from all
18 patients was determined according to a commercial assay at the
time of admission to the emergency room to be <350 pg/ml (10%
cutpoint), and 12 were <100 pg/ml (99th %) percentile. These
samples were tested at later times using the same commercial assay,
and were determined to test positive for cTnI. The same serum
samples were also assayed for TnI according to the assay of the
invention as described in Examples 1 and 3, and the results
compared to the results obtained using the commercial assay.
[0302] Blood was drawn for the first time at the time the patient
presented with chest pain (sample 1), and subsequently at intervals
between 4-8 hours (samples 2 at 12 hours; sample 3 at 16 hours;
sample 4 at 24 hours; sample 5 at 30 hours; sample 6 at 36 hours;
sample 7 at 42 hours; and sample 8 at 48 hours). The serum was
analyzed by the methods of the invention and by a current
commercial method, and the results obtained are shown in FIG. 7.
The analyzer of the invention detected TnI at the time the patient
presented with chest pain (sample 1), while the commercial assay
first detected cTnI at a much later time (sample 6 at 36 hours).
The concentration of TnI in sample 3 exceeded the biological
threshold level that was established using the analyzer of the
invention (7 pg/ml, see FIG. 5), and indicated that sample 3 is
positive for TnI to suggest the incidence of a cardiac event. The
biological threshold for the commercial assay lies between 111 and
333 pg/ml of TnI. Accordingly, sample 3 would not have been
considered to indicate a possible cardiac event.
[0303] In addition, the methods and compositions of the present
invention allow for much earlier diagnosis and possible
intervention based on cardiac troponin levels, as evidenced by
results for the first sample taken from the patients. In the 3
cases that had initial commercial assay cTnI values of between 100
and 350 ng/ml, all were positive for cTnI by the analytical methods
of the invention (i.e., cTnI over 7 pg/ml). In the 12 cases that
had initial commercial cTnI values of less than 100 pg/ml, 5 were
determined to be positive for a cardiovascular event according to
the assay of the invention (i.e., cTnI over 7 pg/ml). The
prospective use of the assay of the invention would have detected
53% more AMI cases than the current commercial assay when the
admission sample was tested.
[0304] Study 2: 50 additional serum samples, which tested negative
according to the commercial assay, were tested using the analyzer
and assay of the invention. The results are shown in FIG. 8. Of the
50 samples, 36 were within the 99th % and determined to be within
the normal range established by the assay of the invention.
However, the remaining 14 samples that were determined to be within
the commercial "normal" or non-diseased range, tested above the
biological threshold established by the invention.
[0305] Therefore, the high sensitivity cTnI assay of the invention
allows for the detection of myocardial damage in patients when cTnI
serum levels are below threshold values by commercially available
technology. The use of the highly sensitive and precise cTnI assay
of the invention enables detection of AMI earlier than with
existing cTnI assays, and thereby provides the opportunity for
appropriate diagnosis and early medical intervention to improve the
outcome.
Example 5
Sandwich Bead-Based Assays for TnI
[0306] The single particle analyzer system is compatible with
assays done in solution using microparticles or beads to achieve
separation of bound from unbound entities, e.g., in the analysis of
cardiac troponin I.
[0307] Materials: MyOne Streptavidin C1 microparticles (MPs) are
obtained from Dynal (650.01-03, 10 mg/ml stock). Buffers use in the
assay include: 10.times. borate buffer saline Triton Buffer (BBST)
(1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3);
assay buffer (2 mg/ml normal goat IgG, 2 mg/ml normal mouse IgG,
and 0.2 mg/ml MAB-33-IgG-Polymer in 0.1 M Tris (pH 8.1), 0.025 M
EDTA, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100, and 0.1% NaN3,
stored at 4 C); sand elution buffer (BBS with 4 M urea, 0.02%
Triton X-100, and 0.001% BSA, stored at 2-8 C). Antibodies used in
the sandwich bead-based assay include: Bio-Ab (A34650228P
(BiosPacific) with 1-2 biotins per IgG) and Det-Ab (G-1129-C
(BiosPacific) conjugated to A647, 2-4 fluors per IgG). The standard
is recombinant human cardiac troponin I (BiosPacific, cat
#J34120352). The calibrator diluent is 30 mg/ml BSA in TBS
wEDTA.
[0308] Microparticles Coating: 100 ul of the MPs stock is placed in
an eppendorf tube. The MPs are washed three times with 100 ul of
BBST wash buffer by applying a magnet, removing the supernatant,
removing the magnet, and resuspending in wash buffer. After the
washes the MPs are resuspended in 100 ul of assay buffer and 15 ug
of Bio-Ab are added. The mixture is then incubated for an hour at
room temperature with constant mixing. The MPs are washed five
times with 1 ml wash buffer as described above. After the washes
the MPs are resuspended in 15 ml of assay buffer (or 100 ul to
store at 4 0C.).
[0309] Preparation of Standard and Samples: The standard is diluted
with calibrator diluent to prepare proper standard curve (usually
200 pg/ml down to 0.1 pg/ml). Frozen serum and plasma samples need
to be centrifuged 10 minutes at room temperature at 13 K rpm.
Clarified serum/plasma is removed carefully to avoid taking any
possible pellets or floaters and put into fresh tubes. 50 ul of
each standard or sample is pipetted into appropriate wells.
[0310] Capture Target: 150 ul of MPs (after resuspension to 15 ml
in assay buffer+400 mM NaCl) are added to each well. The mixture is
incubated on JitterBug, 5 at room temperature for 1 hr.
[0311] Washes and Detection: The plate is placed on a magnet and
the supernatant is removed after ensuring that all MPs are captured
by the magnet. 250 ul of wash buffer are added after removing the
plate from the magnet. The plate is then placed on the magnet and
the supernatant is removed after ensuring that all MPs are captured
by the magnet. 20 ul Det-Ab are added per well (Det-Ab to 500 ng/ml
is diluted in assay buffer+400 mM NaCl)). The mixture is incubated
on JitterBug, 5 at room temperature for 30 min.
[0312] Washes and Elution: The plate is placed on a magnet and
washed three times with wash buffer. The supernatant is removed
after ensuring that all MPs are captured by the magnet and 250 ul
of wash buffer are added. After the washes the samples are
transferred into a new 96-well plate. The new plate is then placed
on the magnet and the supernatant is removed after ensuring that
all MPs are captured by the magnet. 250 ul of wash buffer are then
added after removing the plate from the magnet. The plate is then
placed on the magnet and the supernatant is removed after ensuring
that all MPs are captured by the magnet. 20 ul of elution buffer
are then added and the mixture is incubated on JitterBug, 5 at room
temperature for 30 min.
[0313] Filter out MPs and transfer to 384-well plate: The standard
and samples are transferred into a 384-well filter plate placed on
top of a 384-well assay plate. The plate is then centrifuged at
room temperature at 3000 rpm with a plate rotor. The filter plate
is removed and the appropriate calibrators are added. The plate is
covered and is ready to be run on SMD.
[0314] SMD: An aliquot is pumped into the analyzer.
Individually-labeled antibodies are measured during capillary flow
by setting the interrogation volume such that the emission of only
1 fluorescent molecule is detected in a defined space following
laser excitation. With each signal representing a digital event,
this configuration enables extremely high analytical sensitivities.
Total fluorescent signal is determined as a sum of the individual
digital events. Each molecule counted is a positive data point with
hundreds to thousands of DMC events/sample. The limit of detection
the cTnI assay of the invention is determined by the mean +3 SD
method.
Example 6
Cross-Reacting Antibodies
[0315] Cross-reacting antibody pairs were prepared for
cross-reactivity with human, monkey, dog, and rat. Assays were
performed on serum samples using a protocol essentially as
described in Example 5, i.e., capture antibody was immobilized on
paramagnetic beads. Results are shown in FIGS. 9 and 10. This
Example demonstrates that antibodies that cross-react to several
species may be produced and used to measure cardiac troponin I at
very low limits of detection, i.e., less than 1 pg/ml across human,
monkey, dog, and rat.
Example 7
Accurate and Reproducible IA-Based Technology
[0316] This example describes a IA-based technology that can
accurately and reproducibly measure proteins with a sensitivity of
10-100 femtogram (fg)/ml with a dynamic range of >4.5 logs in
sample volumes of 50-150 ul.
Materials and Methods
[0317] Materials
[0318] Antibodies and analytes (recombinant) were obtained from
R&D Systems (Minneapolis, Minn). Fluorescent dyes and biotin
succimidyl ester, used to label antibodies, were obtained from
Invirtogen (San Diego, Calif.). Rat, dog, and monkey cTnI were
purified from natural sources and obtained from Hytest (Sweden).
Human lithium citrate plasma specimens were purchased from
Interstate blood bank. Streptavidin coated paramagnetic
microparticles (MPs) were obtained from Invitrogen. Antibodies were
labeled with fluorescent dye (detection antibody, usually
polyclonal) and biotin (capture antibody, usually monoclonal) using
manufacturers recommendations. MPs were coated with biotinylated
antibody under saturation conditions, washed and stored in assay
buffer (1% BSA based buffer containing non-ionic detergent and
heterophile/HAMA antibody blocking reagents).
[0319] Immunoassays
[0320] Unless stated otherwise, the typical immunoassay was
performed as follows. Samples or standards (e.g. 50 ul-100 ul) were
diluted with assay buffer containing capture antibody coated MPs
(e.g. 150 ul) and incubated in a 96 well plate for one to two hours
at 25.degree. C. with shaking. MPs were separated using a magnetic
bed (Ambion, Tex.). Supernatant was removed and then 20 ul of
detection antibody was added and incubated for 60 minutes at
25.degree. C. with shaking. The MPs were again magnetically
separated and washed six times using buffered saline plus
surfactant. After removal of residual wash buffer, 20 ul of elution
buffer was added. This reagent disrupted antibody-analyte
interactions and resulted in the release of detection antibody from
the MPs. The solution in each 96-well was then transferred to a
384-well filter plate and centrifuged at 3,000 RPM for three
minutes to separate detection antibody in elution buffer from MPs.
The eluted and filtered material in the 384-well plate was then
placed into the analyzer system.
[0321] Analyzer System
[0322] The analyzer system is based upon single molecule counting
technology. Liquid is sipped well and pumped through a capillary
with a 100 micro-meter diameter. The liquid passes through a
interrogation space comprising a laser for fluorescent dye
excitation and fluorescent emission is detected via confocal
microscopy. The 4.5 plus log dynamic reporting range was obtained
by using a combination of output signals. At low analyte
concentrations, individual fluorescently-tagged detection antibody
molecules were counted as they passed through the interrogation
space in the instrument's flow cell. This signal is a sum of all
such measured events over a set time interval and is termed
detected events (DE). At higher concentrations of analyte, multiple
molecules were detected simultaneously in the flow, and signal was
measured as photon events (PE) over the time interval. At this
point, total light measured during the time event was used. At the
highest concentrations of analyte, total photons (TP), the sum of
all photon events, was used. DE, PE and TP signal were used to
generate a weighted four-parameter curve fit for each signal type.
Finally, a computer algorithm was created that combined these three
curve fits into a weighted single curve fit that provided a >4.5
log linear reporting range.
Results
[0323] Reporting Range, Linearity, Accuracy and Reproducibility
[0324] To determine the reporting range for the IL-6 assay,
recombinant human IL-6 was serially diluted (1 ng/ml to 10 fg/ml)
in a goat serum-based diluent. Samples of 100 ul were run in
quadruplicate. The reporting range of a typical bioassay is
presented in FIG. 11. Resulting signal was back interpolated using
the curve fit algorithm. A linear response (R.sup.2=0.99;
y=0.97+6.4) was observed from 10 fg/ml to 400 pg/ml, representing
an approximate 4.5 log reporting range (FIG. 11). Accuracy was
determined by measuring spike recovery of analyte (IL-6 and human
cardiac troponin-I, cTnI) into panels of human plasma. The IL-6 and
cTnI assays and spike levels of 5 and 50 pg/ml, average %
recoveries were between 90% and 110% for both analytes at both
concentrations (data not shown). Results from performing the cTnI
immunoassay over eight consecutive runs and back interpolating the
standard curves are presented in FIG. 12. In these experiments, the
highest concentration standard used was 100 pg/ml. A linear
(R.sup.2=0.99) response was observed at both the high and low ends
of the standard curve shown. The % CV for the 8 assay runs for back
interpolated determinations was <10% between for all values
>0.78 pg/ml. The % CV was 16% and 23% for the 0.39 and 0.2 pg/ml
values, respectively.
[0325] Sensitivity
[0326] Sensitivity or LoD for the analyzer, composition, and
methods of the immunoassay system varied from analyte to analyte.
Table 6 depicts the LoD for 10 different human immunoassays. Larger
sample volumes consistently resulted in lower LoDs and enhanced
sensitivity. For example, sample volumes >50 ul resulted in LoDs
ranging from 0.01 pg/ml (human IL-6) to 0.12 pg/ml (human VEGF). As
sample volumes decreased, LoD's increased in a proportional manner.
All of the assays were performed in a two-step manner with 1-2
hours of capture and 1 hour of detection.
TABLE-US-00007 TABLE 6 The effect of sample volume on immunoassay
sensitivity (LoD) Volume LoD Analyte (ul) LoD pg/ml Volume (ul)
pg/ml MCP-1 200 0.03 20 0.25 RANTES 100 0.05 20 0.3 VEGF 100 0.12
20 1.0 IL-8 50 0.12 20 0.3 IL-1a 200 0.01 20 0.1 IL-7 200 0.02 10
0.3 IL-6 100 0.01 10 0.15 TNF-a 200 0.02 10 0.4 IL-1B 150 0.02 10
0.3 cTPN-I 100 0.11 10 1.2
[0327] The effect of incubation time on assay sensitivity is shown
in Table 7. In this case a single step cTnI assay was performed
using 50 ul of sample and simultaneous capture and detection
reactions. As incubation times, increased assay sensitivity
improved. This was due to an improvement in slope response as a
function of time. Of note, even with a 15 minute incubation a LoD
of 0.61 pg/ml was achieved (using the NIST reference material).
TABLE-US-00008 TABLE 7 The effect of incubation time on a one step
cTnI immunoassay sensitivity (LoD) Time (minutes) Background Slope
LoD pg/ml 120 184 152 0.241 60 172 102 0.336 30 173 90 0.383 15 193
64 0.61
[0328] To assess species cross reactivity, antibodies were chosen
for the cTnI bioassay that should cross react across these species
based upon cTnI homology at reactive epitopes. Serial dilutions of
individual cTnIs from human (NIST material), rat, dog and monkey
were made in a BSA-based diluent. Sample volumes of 50 ul were then
tested with the analyzer system. The findings are presented in
Table 8. Slopes of the dilution curves ranged from 68-138, and LoDs
ranged 0.14-0.72 pg/ml. Bias against the human NIST standard was
1.6, 2.0, and 4.9 for rat, dog and monkey, respectively. The reason
for this bias is not clear but may reflect inconsistencies in
analyte value assignment by the manufacturer. Regardless all 4
species cTnI provided immunoassays with LoDs <1 pg/ml.
TABLE-US-00009 TABLE 8 Species cross reactivity of the analyzer
system cTnI immunoassay. Species of cTnI Background Slope LoD
(pg/ml) Human (NIST) 143 138 0.19 Monkey 147 28 0.72 Dog 138 68
0.58 Rat 138 84 0.14
[0329] The sensitivity of the human cTnI assay was employed to
define the range of cTnI in human plasma obtained from 100 human
blood donors (50 ul sample size). The results are presented in FIG.
13. The values ranged from <2 pg/ml to 39 pg/ml with an average
value of 2.19 pg/ml (4.1SD). Three plasma had values <0.2 pg/ml
which was the LoD. The value for the 99.sup.th percentile was 9
pg/ml. If the one apparent outlier of 39 pg/ml was removed from the
data set the mean for the population was 1.83 pg/ml (1.9SD) with
the 99.sup.th percentile at 8 pg/ml.
[0330] This example shows the combination of ultra-sensitive flow
detection with MP based IA technology provides protein bioassays
with analytical LODs in the fg/ml range, which is approximately
10-fold more sensitive than previously demonstrated. We have
applied this technology to create important bioassays for
clinically useful protein biomarkers, such as cTnI and IL-6. This
new development in IA technology provides accurate, sensitive and
reproducible quantitative bioassays with a dynamic range of
measurement in small sample volumes across multiple mammalian
species.
[0331] The analyzer, compositions, and methods provide enhanced
sensitivity via two mechanisms. First, unlike analog systems that
count total light (e.g. a calorimetric 96-well plate reader), the
analyzer system separates background fluorescent signal from
antibody-tagged signal by thresholding out the background signal
and measuring each fluorescently-labeled detection antibody as a
digital event (Wu et al). Second, the MP solid phase has been
designed to minimize non-specific binding (NSB) of
fluorescently-labeled detection antibodies. Furthermore,
incubations are performed in 96-well plates with polypropylene
wells that have very low protein binding capacity and, thus, low
NSB. With the analyzer system, when a fluorescently-tagged
detection antibody is released from the MPs, the level of NSB in
reactions that contain no analyte is very low. These
characteristics have allowed us to use relatively high
concentrations of detection antibody (100-500 ng/ml) which favor a
steep assay response while maintaining a very low background.
[0332] The sensitivity of the analyzer, compositions, and methods
allows for the measurement and quantification of biomarkers in
normal, non-disease states. This is an important foundation that
must be established before changes in biomarkers can be used to
diagnose disease and monitor progression and therapeutic
interventions. We quantified the levels of cTnI in a panel of
lithium heparin plasma obtained from 100 human blood donors. The
mean and range of cTnI values in this plasma are similar to those
reported previously for serum specimens. In the case of cTnI, we
demonstrated an analytical LoD of 0.2 pg/ml and a 10% CV cutpoint
between 0.78 and 1.6 pg/ml. Since normal subjects have serum and
plasma cTnI levels between 0.3-9 pg/ml (average 2-3 pg/ml),
increases from these normal levels can be measured with a high
level of precision in most human serum and plasma samples. We have
also shown that the analyzer, compositions, and methods provide
analytical sensitivities <1 pg/ml across multiple mammalian
species that are often used in preclinical studies, such as monkey,
rat and dog. This finding may have significant applications in drug
development where the monitoring of cardiac toxicity in preclinical
animal models is desired.
[0333] The enhanced sensitivity that analyzer, compositions, and
methods provide has additional benefits; namely sample volume
requirements and incubation times. For example, to achieve highest
levels of sensitivity, often sample volume is increased in IA
applications. By providing a more some sensitive approach, less
sample volume can be used with the analyzer system thus enabling
the preservation of precious specimens. One of the limitations of
rapid tests used today near patient settings is a lack of
sensitivity compared to automated system counterparts. This is
attributable to technology, sample volume, as well as incubation
times. We demonstrated a proportional increase in sensitivity with
incubation times. The analyzer, compositions, and methods have
sensitivities of 10-100 fg/ml using 100 ul of sample and one to two
hours incubation. Using a one-step modification for the analyzer
system as described above we were able to reduce the incubation
times to 15 minutes with an approximate LoD of 0.6 pg/ml with 50 ul
of sample. This is 50-200 times more sensitive than a variety of
commercially available cTnI assays on automated platforms. Taken
together these data demonstrate the potential for the development
of a clinically useful rapid sub pg/ml IA using single molecule
counting as incorporated in the analyzer system
[0334] Approaches to enhance ELISA sensitivity have involved the
use of a variety of technologies including signal amplification
(e.g. double enzymatic amplification). Although high sensitivity
IAs have been demonstrated using this approach, often the magnitude
of assay dynamic range is compromised to a 1.5-2.0 log range. If
the analyte of interest exists in a wide biological range, such as
for cytokines, then a sample will often require numerous dilutions
to achieve the correct concentration for measurement in the IA.
[0335] The analyzer system builds single molecule detection
combined with MP IA technology to provide highly sensitive
immunoassays. It also provides a broad dynamic reporting range and
flexible sample volume usage. Dynamic or broad reporting range is
important in the ability to detect both normal and disease states
in one standardized bioassay. The flexibility of sample volume
usage can be used to determine multiple quantifications from a
small sample or to conserve samples. If ultimate sensitivity is not
required, for example if the analyte is in the 20-100 pg/ml range,
multiple analyses can be made. In the case where the normal range
of a protein biomarker is in the low 10-20 pg/ml range, a highly
sensitive assay could be combined with small sample requirements to
enable sample conservation and potential usage for the measurement
of additional biomarkers. This could be especially important when
analyzing rodent samples or human repository specimens.
Example 8
Short and Long-Term Biological Variation for Cardiac Troponin I
Using a High Sensitivity Assay: Implications for Clinical
Practice
[0336] As serial testing will be important in the interpretation of
low concentrations, we determined the short-term (4-hours) and
long-term (8-weeks) biological variability, reference change values
and index of individuality for cTnI. Assays for cardiac troponin
have been the mainstay for diagnosis of acute myocardial infarction
(AMI) and risk stratification for future adverse cardiac events. In
the clinical context of myocardial ischemia, the European
Society/American College of Cardiology have redefined AMI to be
predicated on an increase in cardiac troponin above the 99.sup.th
percentile of a healthy population with a imprecision of 10% or
less. Up until recently, there have been very few commercial assays
that can detect cardiac troponin I (cTnI) concentrations in healthy
subjects with the requisite precision. Recently, a new high
sensitivity troponin assay was developed using single photon
fluorescence detection. In preliminary studies, this assay had a
sensitivity of 0.2 ng/L, and a 10% coefficient of variance (CV) at
1.8 ng/L. The 99.sup.th percentile cutoff concentration obtained
from a healthy population was lower at 10 ng/L than any of the
other commercial assays for cTnI.
[0337] The clinical need for very high sensitivity troponin has
been recently reviewed. There are three major areas where
next-generation cTnI assays can potentially improve current
practice: earlier diagnosis than is currently possible with
existing routine markers (troponin, Creatine kinase-MB, and
myoglobin), improved risk stratification for future adverse cardiac
events among AMI ruleouts, and monitoring of therapeutic drugs that
have the potential to cause cardiotoxicity. For AMI patients, the
first detectable increase in troponin after the onset of chest pain
will enable an earlier diagnosis and triage of patients to the
appropriate level of cardiac care. Melanson et al. showed that use
of a high-sensitivity cTnI assay was positive earlier than use of a
lower sensitivity assay 64% of confirmed AMI cases. For risk
stratification, use of the lowest troponin cutoff (i.e., 99.sup.th
percentile) in the TIMI-TACTICS II trial identified 12% more cases
of patients who were subsequently determined to be at high risk for
future AMI and cardiac deaths. For cardiotoxicity, chemotherapeutic
drugs such as the anthracyclines release small amounts of troponin
as the result of injury to the heart. Use of a high-sensitivity
troponin assay may enable detection of other drugs that are
cardiotoxicity (e.g., cyclooxygenase-2 inhibitors), where existing
assays lack the requisite analytical sensitivity.
[0338] Increasing the analytical sensitivity will result in a
lowering of the cutoff concentration at the 99.sup.th percentile,
and will result in a higher frequency of positive troponin results
in patients who present to the emergency department with chest
pain. Unless ischemia can be ruled out by clinical or other means,
a mildly increased troponin will produce more false positive cases
of AMI. In order to reduce the diagnostic confusion caused by high
sensitivity troponin assays, the National Academy of Clinical
Biochemistry advocated the use of serial testing. Troponin
concentrations that are stable over an appropriate sampling
interval are more likely caused by chronic diseases such as renal
failure, heart failure, sepsis, and myocarditis. The NACB
recommended a change of 20% from baseline as suggestive of an AMI
that is either evolving (rise in troponin) or resolving (fall in
troponin). This limit was determined based a calculation of three
times the imprecision at the cutoff concentrations and not from the
marker's biological variation. There have been no recommendations
to date as to how serial results of troponin should be interpreted
in the context of risk stratification for future adverse events or
cardiac toxicity due to therapeutic drugs.
[0339] A more rigorous determination of statistically significant
reference change values of serial testing is based on measurement
of an analyte's biologic variation. These studies are usually
conducted in healthy subjects with no evidence of active
cardiovascular disease. However, until assays were available that
could reliably detect troponin in healthy subjects, such studies
were not possible. Biological variability studies cannot be
conducted on diseased patients due to the changing concentrations
of troponin over time in patients with acute coronary syndromes
(ACS) or chronic diseases such as heart and renal failure.
Therefore, we determined the short- and long-term biological
variability on healthy subjects using a prototype cTnI assays with
10-50 times higher analytical sensitivity than the best that is
available among the commercially-available assays.
Materials and Methods
[0340] Patients and Specimens
[0341] Two separate cohorts of subjects were recruited for this
study using a protocol that was reviewed and approved by the
University of California Committee for Human Research and the San
Francisco General Hospital (SFGH) General Clinical Research Center
(GCRC). All subjects signed a written consent. For the short-term
biological variability, 12 subjects (6 males and 6 females, age
range 23-54 y) were recruited. A heparin lock was inserted into the
antecubital vein by staff at the SFGH GCRC. Thereafter, blood was
collected at 0, 1, 2, 3, and 4 hours through the heparin lock into
vacutainer tubes containing no anticoagulant. A small amount of
blood was discarded prior to blood collection to flush the line of
heparin. After collection, a small amount of heparin was injected
into the intravenous lines to maintain patency. The collected blood
was allowed to clot, the tubes centrifuged within 30 minutes, and
the serum aliquoted and frozen at -70.degree. C. until analysis.
For the long-term biological variability, 17 subjects (9 males and
10 females, age range 19-58 y) were recruited. Blood was collected
through venipuncture every other week for 6-8 weeks (3-4 total
samples per subject). Samples were centrifuged and stored at
-70.degree. C. and thawed (once) prior to analysis. To minimize the
pre-analytical variability, the same phlebotomist was used for each
subject. For the long-term study, blood was collected on the same
day of the week and roughly the same time of the day. For both
cohorts, the subjects self-reported no symptoms or history of heart
disease.
[0342] Data Analysis
[0343] Frozen samples were allowed to thaw at room temperature. All
analytical assays for cardiac troponin were performed at Singulex
Inc., Hayward, Calif. using the analyzer, compositions, and
methods, and were tested in duplicate using the same technologist
on the same day (different days for each of the short- and
long-term studies). All results and calculations were performed
using Microsoft Excel (Redmond, Wash.). We use the Cochran's test
to determine the presence of outliers among patient results. We
found that the distribution of troponin data was non-parametric and
therefore performed a log normal transformation of the troponin
results prior to calculation of the biological variation. The
equations established by Fokkema et al. were used instead of the
more widely-used nested ANOVA procedures of Fraser et al., who do
not describe log transformation. For calculation of the reference
change value, this transformation resulted in nonsymmetric limits,
i.e., separate criteria for statistically significant difference
between increasing and decreasing troponin values. We determined
the goals for precision (.ltoreq.0.5.times.CV.sub.I) and accuracy
(.ltoreq.0.25.times.(CV.sub.I.sup.2+CV.sub.T.sup.2).sup.1/2) using
the convention of Fraser et al.
[0344] In summary, for the assessment of short-term variation,
blood was collected on every hour for 4 hours (5 samples total
each) on 12 healthy subjects. For the assessment of long-term
variation, blood was collected every other week for 8 weeks (3-4
samples each) on 17 healthy subjects. Blood was tested using a high
sensitivity cTnI assay (limit of detection 0.2 ng/L). The
analytical coefficient of variance (CV.sub.A), intra-individual
(CV.sub.I) and total median (CV.sub.T) variation, index of
individuality were computed. As the data was non-parametrically
distributed, the reference change values (RCV) were calculated
using a log normal approach.
Results
[0345] The distribution of results for the short and long-term
biological variation for cardiac troponin is shown in FIGS. 14A and
14B, respectively. The mean (points) and range (bars) of values are
given. The data shown in FIG. 14A is the short-term biological
variation on 12 subjects over 4 hours. The data shown in FIG. 14B
is the long-term variation on 15 subjects over 8 weeks. In both
FIGS. 14A and 14B, statistical outliers were removed. There were no
outliers from the hour-to-hour dataset. From the week-to-week
dataset, we determined one subject was determined to be an outlier
and was removed. None of the points had values that were below the
detection limit of the assay of 0.2 ng/L, and all were below the
99.sup.th percentile limit of 10 ng/L, previously determined on 100
apparently healthy subjects. Table 9 shows the results of the short
and long-term biological variability for cardiac troponin.
TABLE-US-00010 TABLE 9 Short and long-term biological variability
(BV) for cTnI PARAMETER SHORT-TERM BV.sup.A LONG-TERM BV.sup.B
Analytical variation CV.sub.A, %7.6 12.2 Biological variation
CV.sub.I, % 7.8 9.0 CV.sub.T, % 10.9 15.2 Index of individuality
0.72 0.59 Precision goals, % 3.9 4.5 Inaccuracy goals, % 3.4 5.2
RCV: lognormal increase 35 52 RCV: lognormal decrease -26 -34
.sup.AOver 4 hours. .sup.BOver 8 weeks.
The values CV.sub.I, and CV.sub.T were slightly lower for the
short-term relative to the long-term. This is expected given that
changes to the heart are not expected to change in a healthy
subject from hour to hour, but may change slightly from week to
week. Goals for imprecision, inaccuracy, and index of individuality
are also shown with the results of the hour-to-hour values being
slightly higher but not significantly different from the
week-to-week results.
[0346] The distribution of the hour-to-hour and day-to-day cTnI
results were non-parametric and right skewed and a log normal
transformation was performed prior to the calculation of RCV. This
resulted in the production of non-symmetrical RCV results, with
higher limits signifying statistical change for increasing cTnI
results and lower limits for decreasing results. The higher RCV
limit for a rising troponin reflects the right skewed distribution
of results from healthy subjects and the greater statistical
uncertainty when values are increasing. The calculated RCV results
are about 1.5 to 2 times higher than the single limit of 20% change
in serial results that was recommended by the NACB. This is partly
due to the higher imprecision observed at the lower cTnI
concentrations observed for healthy subjects.
[0347] The short-term CV.sub.A, CV.sub.I and CV.sub.T for cTnI were
7.6% (mean 2.3 ng/L), 7.8%, and 10.9%. Corresponding results for
the long-term variation were: 12.2%, 9.0%, and 15.2%. The index of
individuality were 0.72 and 0.59, respectively. The RCV were 35%
(increase) and -26% (decrease) for short-term, and 52% and -34%,
respectively with 95% confidence. The low index of individuality
indicates that a population-based reference intervals are less
useful than measuring serial change values in individual patients.
This is particularly important for the interpretation of patients
who have low concentration increases in cTnI using very
high-sensitivity assays for patients presenting with chest pain
(short-term) and for evaluation of drugs for cardiotoxicity
(long-term).
[0348] The determination of the biological variation for cardiac
troponin has not been previously possible due to the lack of
analytical sensitivity of assays to reliably measure troponin in
the blood of healthy subjects. The availability of high sensitivity
troponin assays now enables the reliable detection of troponin due
to the normal turnover of cardiac myocytes with the requisite
imprecision (.ltoreq.10%). Two different research assays have now
shown that there is anon-parametric distribution of cardiac
troponin in healthy subjects. The results in this study were
consistent with these previous findings.
[0349] Table 10 compares the biological variation for cardiac
troponin when measured over the short term, against the other
commonly used cardiac biomarkers of ACS. Total CK, CK-MB activity,
CK-MB mass, and myoglobin are described as "event markers," such as
plaque erosion in the case of AMI. The CV.sub.I for these troponin
and CK-MK mass are low relative to the CV.sub.T, producing a low
index of individuality. An low index (<0.6) indicates that
reference intervals will be of little utility for interpretation of
individual results whereas an index that his high (>1.4)
indicates that reference values are of significant value. Table 10
shows that the traditional ACS markers have a low index. In this
study, the short-term troponin index was slightly higher at 0.72
than the other markers, reinforcing the notion that serial testing
is an important criteria for diagnosis of low level increases.
[0350] The biological variation data does not influence the
interpretation of patients who have a very high troponin
concentration, e.g., a 10-fold increases relative to the 99.sup.th
percentile cutoff. These patients should be immediately admitted
and treated without a need for serial troponin testing. In
contrast, proper use of the biological variation will be important
in the interpretation of minor increases of troponin, i.e., values
at or just above the 99.sup.th percentile limit when very high
sensitivity assays are used. Serial cardiac troponin values that
exceed the upper RCV in the clinical context of chest pain increase
the likelihood that the patient is suffering an evolving AMI. This
interpretation may be valid even if all serial troponin results are
below the population-based reference limit (due to the low index of
individuality). However, there may be other acute disease processes
that can increase troponin concentrations over the short term, such
as sepsis. Patients with serial troponin values that are mildly
increased but are unchanging would increase the likelihood that a
chronic cardiac condition is present that is known to cause cardiac
damage (e.g., myocarditis, heart and kidney failure). Patients with
declining serial troponin results might indicate a resolving AMI,
particularly if there is a history of chest pain from the prior
days or week. It should be noted that patients with a positive
troponin due to a chronic disease can present with an acute
exacerbation and increasing troponin values that mimic an AMI
release pattern. Therefore clinical judgment remains paramount in
the interpretation of troponin testing.
[0351] Table 10 also compares long-term biological variation of
cardiac troponin against other biomarkers that are used for chronic
disease detection.
TABLE-US-00011 TABLE 10 Summary of Biological Variation for Cardiac
Markers MARKER CV.sub.A CV.sub.I CV.sub.T II RCV DURATION REFERNCE
Acute disease markers Creatine kinase 14.0 22 42.2 0.52 72.2 Daily
Ross et al. CK-MB, activity 29.1 4.9 14.1 0.35 81.8 Daily Ross et
al. CK-MB, mass 6.8 18.4 61.2 0.30 54.4 Daily Ross et al. Myoglobin
13.4 17.6 46.6 0.38 61.2 Daily Ross et al. Troponin I 7.6 7.6 10.9
0.72 +35, -26 Hourly This Study Chronic disease markers Myoglobin
6.0 11.1 13.8 0.80 35.0 Weekly Panteghini et al. C-reactive protein
5.2 42.2 92.5 0.46 118 Weekly Macy et al. C-reactive protein 1.0
36.8 62.2 0.59 102 4 days Cho et al. Serum amyloid A 4.0 25.0 61.0
0.40 70.1 Weekly Melzi d'Eril et al. Myeloperoxidase 4.0 36.0 30.0
1.20 100 Weekly Dednam et al. BNP 8.4 40.0 41.0 0.98 113 Weekly Wu
et al. BNP +198, -66 Weekly Fokkema NT-proBNP 3.0 35.0 35.0 1.00 98
Weekly Bruins et al. NT-proBNP 1.6 33.3 36.5 0.90 92 Weekly Wu et
al. NT-BNP +157, -61 Weekly Fokkema Troponin I 12.2 9.0 15.2 0.59
52, -34 Weekly This Study .sup.1Conducted in healthy subjects.
.sup.2Conducted in stable heart failure patients. .sup.3Lognormal
transformation of Bruins et al. (22) data.
These markers include myoglobin, high sensitivity C-reactive
protein (hsCRP), serum amyloid A, and myeloperoxidase for
atherosclerosis, and B-type natriuretic peptide (BNP) and NT-proBNP
for heart failure 23). Both the CV.sub.I and CV.sub.T are in
general higher for long-term "disease" markers than for the
short-term event markers. This is not unexpected given that the
interval between blood collections is weeks instead of hours. The
low index of individuality for the inflammatory markers (0.4-0.59,
from Table 2) suggests that reference intervals will not be useful,
and that serial testing will be required. In contrast, for the
natriuretic peptides, the higher index of individuality (0.9-1.1,
Table 2) suggests that reference intervals are appropriate. For
both the inflammatory markers and natriuretic peptides, the high
biological variation results in relatively high RCV values (70 to
198%). Therefore minor changes in serial results will not be
meaningful as recently suggested for hsCRP. Others have challenged
the utility of biological variation for determining significant
serial change values for hsCRP and BNP. The results for the index
of individuality for cardiac troponin are between hsCRP and
BNP.
[0352] The results for cardiac troponin for monitoring of cardiac
disease are significantly different than for the other chronic
cardiac disease markers. The biological variation, index of
individuality and RCVs are much lower. Although hsCRP,
myeloperoxidase and BNP are useful for risk stratification, the
results on troponin suggest that high sensitivity assays can be
applied to detection of minor myocardial damage due to drugs that
may be cardiotoxic. Patients on these drugs are currently not
routinely tested for cardiac troponin. This is because the existing
commercial assays lack the sensitivity to low to detect very minor
and chronic increases. With the development of high sensitive
assays, the utility of cardiac troponin should be re-examined for
this application. If proven to be useful, the long-term biological
variation and calculation of the RCV will be relevant. Recently,
high-sensitivity troponin T assays have been developed and similar
biological variability studies need to be performed to determine
how serial changes can be interpreted in the context of acute and
chronic cardiovascular disease.
Example 9
Specificity of a High Sensitivity Cardiac Troponin-I Assay
Utilizing Single Molecule Counting Technology
[0353] Recent guidelines for cTnI recommend a 99.sup.th percentile
cutoff value with assay imprecision <10%. However, most
commercial assays do not meet these criteria and cTnI values below
the 99.sup.th% value provide diagnostic and prognostic
information.
[0354] Values of cardiac troponin I are the mainstay for the
diagnosis or exclusion of acute myocardial infarction (AMI) and for
risk stratification in patients who present with acute coronary
syndromes. The European Society of Cardiology, American Heart
Association, World Heart Federation and American College of
Cardiology have recommended that the 99.sup.th percentile be used
as the most appropriate cutoff concentration for troponin. It is
recommended that there be no more than 10% assay imprecision at the
99.sup.th percentile value. Given that most commercial assays do
not have the sensitivity and precision to meet these goals and the
fact that it has become clear over time that detectable values
below the 99.sup.th percentile value carry important prognostic
information, there is interest among manufacturers to increase the
sensitivity of commercial troponin assays.
[0355] Recently, a new cardiac troponin I (cTnI) assay was
described that uses single-photon fluorescence detection and
antibody coated microparticles (MPs) as a solid phase capture
reagent. It appears considerably more sensitive than commercial
assays. The analyzer, compositions, and methods of the cTnI assay
reports values of cardiac troponin I in the blood of healthy
subjects, with a preliminary 10% CV at 1.8 ng/L and a 99.sup.th
percentile cutoff value of 10 ng/L. These lower cutoff values
likely will produce more and earlier positive test results in
patients who present to emergency departments with chest pain than
the older troponin assays.
[0356] It is important to demonstrate the analytical specificity of
this highly sensitive cTnI assay with regard to false-negatives and
false-positives. Some degree of assay interference has already been
recognized from heparinized and chelated serum samples, as well as
troponin autoantibody interference, which produce false-negative
results at low troponin concentrations. False-positives may occur
if troponin readings from healthy subjects are due to non-specific
binding of serum or plasma components to the antibodies used in the
assay, rendering results of the assay invalid. This investigation
was designed to indirectly test this issue, predicated on the
assumption that if a non-specific cTnI signal was being obtained
from the samples, it would also be observed by other antibody
coated MPs.
Methods
[0357] Concentrations of cTnI were determined from 20 normal
subjects across four specimen types (serum, EDTA, lithium heparin
and citrate plasma) and 8 additional serum samples from apparently
healthy blood donors (AVG+/-SD). To test for specificity,
microparticles (MPs) were coated with a panel of unrelated
antibodies (A-beta42, MIP-1a, G-CSF and PSA) or left un-coated
(blank MPs) and assayed for cTnI. Standard curves were generated
using the NIST cTnI standard analyte compared to a skeletal
troponin analyte.
[0358] Serum, EDTA, lithium heparin and sodium citrated plasma was
collected from 20 subjects who were free of cardiac disease or
symptoms (13 female, 7 male, average 43 y, range 23-64 y). The
protocol was reviewed and approved by the University of California
Committee on Human Research and all subjects signed a written
consent. The blood was centrifuged within 30 minutes of collection,
aliquoted and stored frozen at -70.degree. C. until analysis. Eight
additional serum samples were obtained from healthy blood bank
donors to test for non-specific antibody interactions (4 male, 4
female, average 25.5 y, range 22-30 y). Frozen serum and plasma
were provided to Singulex (blinded to donor ID) and assayed with
the analyzer, compositions, and methods. Briefly, specimens were
thawed at room temperature, centrifuged for 10 minutes at
13,000.times.g and the clarified specimen was placed into a clean
tube. Each 50 ul specimen (neat serum or plasma) was mixed with 150
ul assay buffer containing paramagnetic microparticles (Dynabeads
MyOne Streptavidin C1, Invitrogen) in the well of a 96 plate. For
analysis across sera types, MPs were coated with a cTnI capture
monoclonal antibody (R&D Systems). For specificity studies, MPs
were either left un-coated (blank MPs) or coated with one of the
following monoclonal antibodies: amyloid-beta-42 (A-beta42,
Covance), macrophage inflammatory protein-1 alpha (MIP-1a, R&D
Systems), granulocyte-colony stimulating factor (G-CSF, R&D
Systems), and prostate specific antigen (PSA, BiosPacific). These
antibodies (both isotype matched and un-matched) were chosen to
represent a variety of antibodies raised against epitopes which are
un-related to cardiac troponin I, to test for evidence of
non-specific antibody interactions in the assay conditions. All
antibodies were biotinylated and coated onto streptavidin MPs at 15
ug IgG per mg of MPs according to manufacturer's recommendations.
The assay consisted of a one hour capture step, one wash of MPs, a
30 minute detection step with polyclonal goat anti cTnI
(Biospacific) labeled with fluorescent dye and diluted in assay
buffer, 5 washes, and 30 minutes of elution of detection antibody
bound to the MPs. All specimens, calibrators and controls were
tested in triplicate and results are presented as an average of the
determinations +/- standard deviation. Inter-assay precision was
determined by assay of two control sera over 14 assay runs
performed over 4 days. Analyte specificity was tested through
linear regression analysis of cTnI standard (NIST) and non-specific
skeletal troponin (Hytest) analytes, assayed over a range of
0.1-100 ng/L.
Results
[0359] cTnI was quantifiable in 93% of serum and plasma specimens,
and no significant differences in cTnI concentrations across
specimen types were observed (95% CI). Out of all samples and
non-specific antibodies tested, one sample displayed quantifiable
non-specific interaction with the A-beta-42 antibody (55% CR). No
non-specific events were observed when skeletal troponin was used
as the analyte.
[0360] From the 20 donor sera and plasma samples collected, there
were no apparent trends in cTnI measurements based upon donor's age
or sex (FIG. 15). Figure shows concentrations of cTnI (ng/L) in
matched serum and plasma specimens obtained from 20 human donors of
mixed age and sex. Samples were run in triplicate and results for
each sample are presented as the mean +/- standard deviation. For
each donor the average cTnI value was assessed across the 4
specimen types (average 3.76+/-4.76 ng/L, range 0.84-22.37 ng/L).
All determinations fell within two standard deviations from the
mean, indicating no significant differences in cTnI concentration
between specimen types (95% CI). One donor presented with elevated
levels of cTnI across all specimen types, and this maximum value
was identified as a statistical outlier using Grub's test
(p<0.05) and removed from further analysis. Removal of this
sample produced an adjusted average cTnI value of 2.78+/-1.93 ng/L
similar to values reported previously. In two control sera,
inter-assay precisions of 7% CV and 10% CV were measured with
average cTnI measurements of 8.3 and 2.2 ng/L, respectively.
[0361] Across all 28 donors, cTnI was detectable in all (LoD 0.2
ng/L) and quantifiable for 93% (LLoQ 1 ng/L) of matched serum and
plasma specimens tested (Table 11).
TABLE-US-00012 TABLE 11 Cross reactivity in normal, human serum
samples of the Singulex MP-based cTnI Assay using non-specific
capture antibodies. Total ND DNQ Quantifiable MP Capture Samples
(<LoD, 0.2 ng/L) (1.0 ng/L > X .gtoreq. 0.2 ng/L)
(.gtoreq.LLoQ, 1.0 ng/L) Antibody (n) (n) % (n) % (n) % cTnI 28 0 0
2 7 26 93 Blank 28 26 93 2 7 0 0 AB-42 20 18 90 1 5 1* 5 MIP-1a 8 6
75 2 25 0 0 PSA 8 7 88 1 13 0 0 G-CSF 8 8 100 0 0 0 0 *One serum
sample displayed 55% CR with AB-42 (1.26 ng/L) compared to cTnI
(2.36 ng/L). Abbreviations: Cross reactivity (CR), non-detectable
(ND), detectable but non-quantifiable (DNQ)
Serum and plasma specimens from one of the twenty volunteers (5%)
was quantifiable for a non-specific antibody, which displayed 55%
cross reactivity with A-beta-42 (1.26 ng/L) compared to cTnI (2.36
ng/L). No other samples were quantifiable for cTnI when using a
non-specific capture antibody or when using blank MPs. Some low
level non-specific reaction (less than the LLoQ and greater than
the LoD) was observed for blank microparticles (7%) and for
non-specific capture antibodies A-beta-42 (5% donors), MIP-1a (25%
donors), and PSA (13% donors). No non-specific reactions were
observed when G-CSF was used as the capture antibody. In tests of
analyte specificity, back-interpolated standard curve analysis of
the NIST standard cTnI analyte (y=1.053.times.-0.229,
R.sup.2=0.999) showed poor correlation when compared to analysis of
the orthologous skeletal troponin analyte (y=0.000.times.+0.156,
R.sup.2=0.001) in the analyzer, compositions, and methods.
[0362] These data support the specificity of the novel single
molecule assay for the quantification of cTnI. We have previously
shown that serum specimens provide a linear response when diluted
with synthetic calibrator diluent, however, we could not exclude
the possibility that with such sensitive limits of detection some
other material was captured along with cTnI and then
non-specifically measured. This study provides a more robust test
by substituting a series of non-specific, non-isotype matched, but
high affinity antibodies in the capture position to further
evaluate that possibility. Our data support the contention that the
assay specifically measures cTnI in most apparently healthy
subjects. Even in subjects that showed low-levels of non-specific
binding, this binding was below the limit of quantification
reported for the assay in all but one case (See Table 11). In the
one case where non-specific binding was quantifiable, it accounted
for 55% of the cTnI signal. The magnitude of this effect would not
have moved this subject out of the normal range projected for the
assay.
[0363] Data like these are critical as the field moves forward. As
more sensitive detection techniques such as the novel assay used in
this approach are developed, confirmation of the specificity of
their detection is essential. Thus, similar cross reactivity
studies which evaluate potential issues with troponin orthologs
(i.e., skeletal muscle TnI and TnT) and other non-specific binding
events are critical adjuncts to the validation of these assays.
Because of the sensitivity of this novel assay, conventional
methods may not be adequate to provide this specificity
information, requiring an equally novel approach to the validation
issue. These data, taken together with those reported previously
predicated on linear dilution and immunodepletion, provide strong
evidence that this particular cTnI assay approach has maintained
the specificity of detection.
[0364] The data presented provides further documentation that the
analyzer, compositions, and methods have high specificity and
reliably measure cTnI in apparently healthy human subjects. This
high degree of specificity and high sensitivity should allow for
further investigation of the clinical significance of mild
increases in cTnI in patients with and without acute coronary
disease.
REFERENCES
[0365] 1. Hogrefe W R. Biomarkers and assessment of vaccine
responses. Biomarkers 2005; 10 Suppl 1:S50-7. [0366] 2. Banks R E.
Measurement of cytokines in clinical samples using immunoassays:
problems and pitfalls. Crit. Rev Clin Lab Sci 2000; 37:131-82.
[0367] 3. Meriggioli M N. Use of immunoassays in neurological
diagnosis and research. Neurol Res 2005; 27:734-40. [0368] 4. Lim C
T, Zhang Y. Bead-based microfluidic immunoassays: the next
generation. Biosens Bioelectron 2007; 22:1197-204. [0369] 5. Martin
K, Viera K, Petr C, Marie N, Eva T. Simultaneous analysis of
cytokines and co-stimulatory molecules concentrations by ELISA
technique and of probabilities of measurable concentrations of
interleukins IL-2, IL-4, IL-5, IL-6, CXCL8 (IL-8), IL-10, IL-13
occurring in plasma of healthy blood donors. Mediators Inflamm
2006; 2006:65237. [0370] 6. Guthrie J W, Hamula C L, Zhang H, Le X
C. Assays for cytokines using aptamers. Methods 2006; 38:324-30.
[0371] 7. Dupuy A M, Lehmann S, Cristol J P. Protein biochip
systems for the clinical laboratory. Clin Chem Lab Med 2005;
43:1291-302. [0372] 8. Dhawan S. Signal amplification systems in
immunoassays: implications for clinical diagnostics. Expert Rev Mol
Diagn 2006; 6:749-60. [0373] 9. Wu A F N, Todd J, Puskas R, Goix P.
Development and preliminary clinical validation of a high sensitive
assay for cardiac troponin using capillary flow (single molecule)
fluorescence detection. Clin Chem 2006; 52:2157-8. [0374] 10.
McGavigan A D, Maxwell P R, Dunn F G. Serological evidence of early
remodeling in high-risk non-ST elevation acute coronary syndromes.
Int J Cardiol 2007. [0375] 11. O'Brien P J. Blood cardiac troponin
in toxic myocardial injury: archetype of a translational safety
biomarker. Expert Rev Mol Diagn 2006; 6:685-702. [0376] 12.
Schupbach J. Viral RNA and p24 antigen as markers of HIV disease
and antiretroviral treatment success. Int Arch Allergy Immunol
2003; 132:196-209. [0377] 13. Toyo-oka T, Kumagai H. Cardiac
troponin levels as a preferable biomarker of myocardial cell
degradation. Adv Exp Med Biol 2007; 592:241-9. [0378] 14. Joint
European Society of Cardiology/American College of Cardiology
Committee. Myocardial infarction redefined--a consensus document of
the joint European Society of Cardiology/American College of
Cardiology Committee for the redefinition of myocardial infarction.
J Am Coll Cardiol 2000; 36:959-69. [0379] 15. Todd J, Freese B, Lu
A, Held D, Morey J, Livingston R, et al. Ultrasensitive flow-based
immunoassays using single-molecule counting. Clin Chem 2007;
53:1990-5. [0380] 16. H B, Fukushima F, Puskas R, Todd J, Goix P.
Development and preliminary clinical validation of a high
sensitivity assay for cardiac troponin using a capillary flow
(single molecule) fluorescence detector. Clin Chem 2006; 52:2157-9.
[0381] 17. H B, Lu A, Freese B, Todd J. Development and preliminary
clinical validation of an ultra-sensitive assay for cardiac
troponin using microparticle based immunoassay and single molecule
counting. Clin Chem 2007; 53(suppl):A15. [Abstract]. [0382] 18. H
B, Jaffe A S. The clinical need for high sensitivity cardiac
troponin assays for ACS and the role for serial testing. Am Heart J
2008; 155:208-14. [0383] 19. Melanson S E F, Morrow D A, Jarolim P.
Earlier detection of myocardial injury in a preliminary evaluation
using a new troponin I assay with improved sensitivity. Am J Clin
Pathol 2007; 128:282-6. [0384] 20. Morrow D A, Cannon C P, Rifai N,
et al. Ability of minor elevations of troponins I and T to predict
benefit from an early invasive strategy in patients with unstable
angina and non-ST elevation myocardial infarction: results from a
randomized trial. JAMA 2001; 286:2405-2412. [0385] 21. Sparano J A,
Wolff A C, Brown D. Troponins for predicting cardiotoxicity from
cancer therapy. Lancet 2000; 356:1947-8. [0386] 22. Levesque L E,
Brophy J M, Zhang B. Time variations in the risk of myocardial
infarction among elderly users of COX-2 inhibitors. CMAJ 2006;
174:1563-9. [0387] 23. Wu A H B, Apple F S, Jaffe A S, Jesse R L,
Morrow D A, Newby K, et al. National Academy of Clinical
Biochemistry Laboratory Medicine Practice Guidelines: use of
cardiac troponin and the natriuretic peptides for etiologies other
than acute coronary syndromes and heart failure. Clin Chem 2007,
53:2086-96. [0388] 24. Cochran W S. The distribution of the largest
of a set of estimated variances as a fraction of their total. Ann
Eugen 1941; 11:47-52. [0389] 25. Fokkema M R, Herrmann Z, Muskiet F
A J, Moecks J. Reference change values for brain natriuretic
peptides revisted. Clin Chem 2006; 52:1602-3. [0390] 26. Fraser C
G, Harris E K. Generation and application of data on biological
variation in clinical chemistry. Crit. Rev Clin Lab Sci 1989;
27:409-37. [0391] 27. Bao P, Wei T F, Cork K, Hollander J, Shipp G.
A novel technology platform for the ultrasensitive detection of
cardiac troponin (cTnI): clinical utility in the emergency
department. 2nd Int Symposium. Integrated Biomarkers in
Cardiovascular Disease. Berlin, June, 2007 [Abstract]. [0392] 28.
Ross S M, Fraser C G. Biological variation of cardiac markers:
analytical and clinical considerations. Ann Clin Biochem 1998;
35:80-4. [0393] 29. Harris E K. Statistical aspects of reference
values in clinical pathology. Prog CLin Pathol 1981; 8:45-66.
[0394] 30. Panteghini M, Pagani F. Biological variation of
myoglobin in serum. Clin Chem 1997; 43:2435. [0395] 31. Macey E M,
Hayes T E, Tracy R P. Variability in the measurement of C-reactive
protein in healthy subjects: implications for reference intervals
and epidemiological applications. Clin Chem 1997; 43:52-8. [0396]
32. Cho L W, Jayagopal V, Kilpatrick E S, Atkin S L. The biological
variation of C-reactive protein in polycystic ovarian syndrome.
Clin Chem 2005; 151:1905-6. [0397] 33. Melzi d'Eril G, Anesi A,
Maggiore M, Leoni V. Biological variation of serum amyloid A in
healthy subjects. Clin Chem 2001; 47:1409-9. [0398] 34. Dednam M,
Vorster B C, Ubbink J B. The biological variation of
myeloperoxidase. Clin Chem 2008, in press. [0399] 35. Bruins S,
Fokkema M R, Pomer J W P, et al. High intraindividual variation of
B-type natriuretic peptide (BNP) and B-type proBNP in patients with
stable chronic heart failure. Clin Chem 2004; 50:2052-2058. [0400]
36. Wu A H B, Smith A C, Mather J F, et al. Biological variation
for NT-pro- and B-type natriuretic peptides and implications for
therapeutic monitoring of patients with congestive heart failure.
Am J Cardiol 2003, 92:628-31. [0401] 37. Wu A H B. Serial testing
of B-type natriuretic peptide and NTpro-BNP for monitoring therapy
of heart failure: the role of biological variation in
interpretation of results. Am Heart J. 2006, 152:828-34. [0402] 38.
Franzini C. Need for correct estimates of biological variation: the
example of C-reactive protein. Clin Chem Lab Med 1998; 36:131-2.
[0403] 39. Clerico A, Zucchelli G C, Pilo A, et al. Clinical
relevance of biological variation of B-type natriuretic peptide.
Clin Chem 2005; 51:925-6. [0404] 40. Thygesen K, Alpert J S, White
H D; Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of
Myocardial Infarction. Universal definition of myocardial
infarction. J Am Coll Cardiol 2007; 50:2173-95. [0405] 41.
Panteghini M, Pagani F, Yeo K T J, Apple F S, Christenson R H, Dati
F, Mair J, Ravkilde J, Wu A H B. Evaluation of the imprecision at
low-range concentration of the assays for cardiac troponin
determination. Clin Chem 2004, 50:327-32. [0406] 42. Schulz O,
Kirpal K, Stein J, Bensch R, Berghoefer G, Schimke I and Jaffe A S.
Importance of low concentrations of cardiac troponins. Clin Chem
2006; 52:1614-5. [0407] 43. Schulz O, Paul-Walter C, Lehmann M,
Abraham K, Berghofer G, Schimke I and Jaffe A S. Usefulness of
detectable levels of troponin, below the 99th percentile of the
normal range, as a clue to the presence of underlying coronary
artery disease. Am J Cardiol 2007; 100:764-9. [0408] 44. Eggers K
M, Lagerqvist B, Venge P, Wallentin L, Lindahl B. Persistent
cardiac troponin I elevation in stabilized patients after an
episode of acute coronary syndrome predicts long-term mortality.
Circulation 2007; 116:1907-14. [0409] 45. Zethelius B, Johnston N,
and Venge P. Troponin I as a predictor of coronary heart disease
and mortality in 70-year-old men: A community-based cohort study.
Circulation 2006; 113:1071-8. [0410] 46. Todd J, Freese B, Lu A,
Held D, Morey J, Livingston R, et al. Ultrasensitive flow-based
immunoassays using single-molecule counting. Clin Chem 2007;
53:1990-5. [0411] 47. Katrukha A, Bereznikova A, Filatov V, Esakova
T. Biochemical factors influencing measurement of cardiac troponin
I in serum [Review]. Clin Chem Lab Med 1999; 37:1091-5. [0412] 48.
Eriksson S, Halenius H, Pulkki K, Hellman J, Pettersson K. Negative
Interference in cardiac troponin I immunoassays by circulating
troponin autoantibodies. Clin Chem 2005; 51:839-47. [0413] 49. Wu A
H B, Fukushima N, Puskas R, Todd J, Goix P. Development and
preliminary validation of a high sensitivity assay for cardiac
troponin using a capillary flow (single molecule) detector. Clin
Chem 2006; 52:2157-8. [0414] 50. Joint European Society of
Cardiology/American College of Cardiology Committee. Myocardial
infarction redefined--a consensus document of the joint European
Society of Cardiology/American College of Cardiology Committee for
the redefinition of myocardial infarction. J Am Coll Cardiol 2000;
36:959-69.
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