U.S. patent application number 11/052219 was filed with the patent office on 2005-08-04 for method and apparatus for increasing the dynamic range and accuracy of binding assays.
Invention is credited to Keys, Daniel A., Reddy, Parameswara M..
Application Number | 20050170409 11/052219 |
Document ID | / |
Family ID | 21866808 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170409 |
Kind Code |
A1 |
Keys, Daniel A. ; et
al. |
August 4, 2005 |
Method and apparatus for increasing the dynamic range and accuracy
of binding assays
Abstract
This invention relates to methods for increasing the dynamic
range and accuracy of assays in which the presence, absence,
activity or concentration of a target analyte is assayed by the
emission or quenching of a light signal, or by a change (i.e., an
evolution or loss) of a light signal in two or more time intervals.
In preferred embodiments multiple digitized images are captured at
varying times, and the images analyzed to identify captured images
within the dynamic range of the assay. The invention further
relates to apparati capable of implementing such methods.
Inventors: |
Keys, Daniel A.; (Irvine,
CA) ; Reddy, Parameswara M.; (Brea, CA) |
Correspondence
Address: |
LINIAK, BERENATO & WHITE, LLC
6550 ROCK SPRING DRIVE
SUITE 240
BETHESDA
MD
20817
US
|
Family ID: |
21866808 |
Appl. No.: |
11/052219 |
Filed: |
February 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11052219 |
Feb 8, 2005 |
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10032790 |
Oct 24, 2001 |
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6867005 |
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Current U.S.
Class: |
435/6.18 ;
435/287.2; 435/6.1; 702/19 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 21/76 20130101 |
Class at
Publication: |
435/006 ;
702/019; 435/287.2 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C12M 001/34 |
Claims
What is claimed is:
1. An apparatus for enhancing the dynamic range of an assay of the
presence, absence, activity or concentration of two or more target
analytes in one or more samples, wherein the presence, absence,
activity or concentration of said target analytes is assayed by the
emission or quenching of a light signal, said apparatus comprising:
(A) one or more containers for receiving a portion of said one or
more samples, said containers additionally containing assay
reagents comprising a compound that, in response to the presence of
a target analyte causes a detectable light signal; and (B) a
computer system comprising a CCD camera detector, said computer
system being specially adapted to detect said light signal and
generate data corresponding to said detected signal; said computer
system additionally processing a capability for comparing said
generated data with data corresponding to the light signal
generated by a known concentration of said target analyte in a
known dynamic range of said assay and report the presence, absence,
activity or concentration of said target analyte; wherein said
computer system causes said CCD camera detector to independently
detect sufficient light signal for each of said target analytes to
ensure that said reported presence, absence, activity or
concentration of each target analyte is determined using data
corresponding to a light signal that is within said known dynamic
range of said assay for that target analyte.
2. An apparatus for enhancing the dynamic range of an assay of the
presence, absence, activity or concentration of two or more target
analytes in one or more samples, wherein the presence, absence,
activity or concentration of said target analytes is assayed by the
emission or quenching of a light signal, said apparatus comprising:
(A) one or more containers for receiving a portion of said one or
more samples, said containers additionally containing assay
reagents comprising a compound that, in response to the presence of
a target analyte causes a detectable light signal; and (B) a
computer system comprising a CCD camera detector, said computer
system being specially adapted to detect said light signal and
generate data corresponding to said detected signal; said computer
system additionally processing a capability for comparing said
generated data with data corresponding to the light signal
generated by a known concentration of said target analyte in a
known dynamic range of said assay and report the presence, absence,
activity or concentration of said target analyte; wherein said
computer system causes said CCD camera detector to independently
detect sufficient light signal for each of said target analytes to
ensure that said reported presence, absence, activity or
concentration of each target analyte is determined using data
corresponding to a light signal that is within said known dynamic
range of said assay for that target analyte; and wherein, for at
least one of said target analytes, said computer system causes said
CCD camera detector to detect light signal cumulatively until a
total detected light signal is obtained that is within the known
dynamic range of said assay for said target analyte; and wherein
said total detected light signal is used to determine said
presence, absence, activity or concentration of said target
analyte.
3. An apparatus for enhancing the dynamic range of an assay of the
presence, absence, activity or concentration of two or more target
analytes in one or more samples, wherein the presence, absence,
activity or concentration of said target analytes is assayed by the
emission or quenching of a light signal, said apparatus comprising:
(A) one or more containers for receiving a portion of said one or
more samples, said containers additionally containing assay
reagents comprising a compound that, in response to the presence of
a target analyte causes a detectable light signal; and (B) a
computer system comprising a CCD camera detector, said computer
system being specially adapted to detect said light signal and
generate data corresponding to said detected signal; said computer
system additionally processing a capability for comparing said
generated data with data corresponding to the light signal
generated by a known concentration of said target analyte in a
known dynamic range of said assay and report the presence, absence,
activity or concentration of said target analyte; wherein said
computer system causes said CCD camera detector to independently
detect sufficient light signal for each of said target analytes to
ensure that said reported presence, absence, activity or
concentration of each target analyte is determined using data
corresponding to a light signal that is within said known dynamic
range of said assay for that target analyte; and wherein, for at
least one of said target analytes, said computer system causes said
CCD camera detector to detect light signal discontinuously at more
than one time interval so that a detected light signal is obtained
that is within the known dynamic range of said assay for said
target analyte; and wherein said detected light signal within the
known dynamic range of said assay for said target analyte is used
to determine said presence, absence, activity or concentration of
said target analyte.
4. The apparatus of claim 3, wherein said computer system stores
the cumulative change in said light signal in two or more of said
time intervals.
5. The apparatus of claim 1, wherein said apparatus simultaneously
assays the presence, absence, activity or concentration of said
more than one target analyte in the same sample.
6. The apparatus of claim 1, wherein said apparatus sequentially
assays the presence, absence, activity or concentration of said
more than one target analyte in the same sample.
7. The method of claim 1, wherein said step (C) is performed
simultaneously for each target analyte being assayed.
8. The method of claim 1, wherein said step (C) is performed
sequentially for each target analyte being assayed.
9. The apparatus of claim 1, wherein said one or more containers is
a multi-well microtiter plate.
10. The apparatus of claim 1, wherein said target analyte has an
activity and wherein said computer system additionally calculates
the rate of a target analyte activity in said sample.
11. The apparatus of claim 1, wherein said target analyte is
selected from the group consisting of an enzyme, a drug or
metabolite, a co-factor, a receptor, a receptor ligand, a hormone,
a cytokine, a blood factor, a virus, an antigen, a steroid, and an
antibody.
12. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of an enzyme.
13. The apparatus of claim 12, wherein said enzyme is selected from
the group consisting of bone specific alkaline phosphatase, aldose
reductase, myoglobin, and troponin I.
14. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of a drug or
metabolite.
15. The apparatus of claim 14, wherein said drug or metabolite is
selected from the group consisting of: an anti-cancer drug,
chemotherapeutic drug, anti-viral drug, non-steroidal
anti-inflammatory drug (NSAID), steroidal anti-inflammatory drug,
anti-fungal drug, detoxifying drug, analgesic, bronchodilator,
anti-bacterial drug, antibiotic drugs, diuretic, digoxin,
anti-metabolite, calcium channel blocker, drug for treatment of
psoriasis, and a substance of abuse.
16. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of a co-factor.
17. The apparatus of claim 16, wherein said co-factor is a vitamin,
T.sub.3, or T.sub.4.
18. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of a cytokine.
19. The apparatus of claim 18, wherein said cytokine is IL-1.beta.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13,
TNF.alpha., VEGF, GMCSF, FGF.beta., INF.gamma., EGF, PDGF, MCSF,
SCF, insulin, VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27,
4-1BB, LNGFR, OX40, TGF.beta.R, or is a ligand of CCR1,
CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4,
BLR1, BLR2, or V28 receptor, or is a ligand of a receptor of IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13.
20. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of a receptor or
receptor ligand.
21. The apparatus of claim 20, wherein said receptor or receptor
ligand is 4-1BB, Axl, BLR1, BLR2, CCR1, CCR2.alpha., .beta., CCR3,
CCR4, CCR5, CD27, CD30, CD4, CD4, CD40, CXCR1, CXCR2, CXCR3, CXCR4,
EGFR, Eph, EPO receptor, Fas receptor, GCSFR, GHR, GMCSFR.alpha.,
gp130, IFNgR.alpha., IFNgR.beta., IFN.alpha.R1, insulin-R,
IL-1.beta., IL-2R .beta., IL-2R.gamma. chains, IL-4R.alpha.,
IL-3R.alpha., IL-5R.alpha., IL-6R.alpha., IL-7R.alpha.,
IL-9R.alpha., IL-10R, IL-11R.alpha., IL-12Rb1, IL-12Rb2,
IL-13R.alpha., GMCSFR.alpha., IL-3/IL-5/GM-CSF receptor common
.beta.-chain, LIFR .beta., LNGFR, MCSFR, Met, OBR, OSMR.beta.,
OX40, PDGFR, PRL, Ron, SCFR, TPOR, TFR, TGF.beta.R, TNFRI, TNFRII,
TPOR, Trk, V28, VEGFR.
22. The apparatus of claim 11, wherein said assay assays the
presence, absence, activity or concentration of a hormone.
23. The apparatus of claim 22, wherein said hormone is adrenaline,
adrenocorticotropic hormone, testosterone, angiotensinogen,
antidiuretic hormone, atrial-natriuretic peptide, calcitonin,
calcitriol, cholecystokinin, chorionic gonadotropin, cortisol,
dopamine, erythropoietin, estradiol, follicle-stimulating hormone,
gastrin, glucagon, gonadotropin-releasing hormone,
gorticotropin-releasing hormone, growth hormone, growth
hormone-releasing hormone, insulin, insulin-like growth factor-1,
leptin, luteinizing hormone, melatonin, aldosterone, neuropeptide
Y, noradrenaline, oxytocin, parathyroid hormone, progesterone,
prolactin, renin, secretin, somatostatin, theophylline,
thiiodothyronine, thrombopoietin, thyroid-stimulating hormone,
thyrotropin-releasing hormone, or thyroxine.
24. The apparatus of claim 11, wherein said assay assays a binding
activity of an antigen or an antibody.
25. The apparatus of claim 24, wherein said assay assays a binding
activity of an antigen characteristic of Chlamydia, Streptococcus
pyogenes Group A bacteria, H. pylori, or M. tuberculosi, hepatitis
virus, rubella, CMV, HIV, FIV, or prostate specific antigen, or an
antibody elicited in response to any of such antigens.
26. The apparatus of claim 11, wherein said assay assays a binding
activity of an autoimmune immunoglobulin, thyroglobulin,
anti-thyroglobulin, IgE, IgG, or IgM immunoglobulin.
27. The apparatus of claim 11, wherein said assay assays a binding
activity of a tumor marker.
28. The apparatus of claim 1, wherein said light signal is an
evolution or loss of a fluorescent light signal.
29. The apparatus of claim 1, wherein said light signal is an
evolution or loss of a chemiluminescent light signal.
30. The apparatus of claim 1, wherein said light signal is an
evolution or loss of an ultraviolet light signal.
31. The apparatus of claim 1, wherein said light signal is an
evolution or loss of a visible wavelength light signal.
32. A method for enhancing the dynamic range of an assay of the
presence, absence, activity or concentration of two or more target
analytes in one or more samples wherein the presence, absence,
activity or concentration of said target analytes is assayed by the
emission or quenching of a light signal, wherein said method
comprises the steps: (A) conducting an assay for the presence,
absence, activity or concentration of each of said target analytes
in said one or more samples wherein said assays each cause light
signals to be emitted or quenched; (B) employing a computer system
comprising a CCD camera detector to detect said light signals, and
to generate data corresponding to said detected signals; and (C)
causing said computer system to compare said generated data using
data corresponding to the light signal generated by a known
concentration of said target analyte in a known dynamic range of
said assay and report the presence, absence, activity or
concentration of said target analyte; wherein said computer system
causes said CCD camera detector to independently detect sufficient
light signal for each of said target analytes to ensure that said
reported presence, absence, activity or concentration of each
target analyte is determined using data corresponding to a light
signal that is within said known dynamic range of said assay for
that target analyte; and wherein, for at least one of said target
analytes, said computer system causes said CCD camera detector to
detect light signal cumulatively until a total detected light
signal is obtained that is within the known dynamic range of said
assay for said target analyte; and wherein said total detected
light signal is used to determine said presence, absence, activity
or concentration of said target analyte.
33. A method for enhancing the dynamic range of an assay of the
presence, absence, activity or concentration of two or more target
analytes in one or more samples wherein the presence, absence,
activity or concentration of said target analytes is assayed by the
emission or quenching of a light signal, wherein said method
comprises the steps: (A) conducting an assay for the presence,
absence, activity or concentration of each of said target analytes
in said one or more samples wherein said assays each cause light
signals to be emitted or quenched; (B) employing a computer system
comprising a CCD camera detector to detect said light signals, and
to generate data corresponding to said detected signals; and (C)
causing said computer system to compare said generated data using
data corresponding to the light signal generated by a known
concentration of said target analyte in a known dynamic range of
said assay and report the presence, absence, activity or
concentration of said target analyte; wherein said computer system
causes said CCD camera detector to independently detect sufficient
light signal for each of said target analytes to ensure that said
reported presence, absence, activity or concentration of each
target analyte is determined using data corresponding to a light
signal that is within said known dynamic range of said assay for
that target analyte; and wherein, for at least one of said target
analytes, said computer system causes said CCD camera detector to
detect light signal discontinuously at more than one time interval
so that a detected light signal is obtained that is within the
known dynamic range of said assay for said target analyte; and
wherein said detected light signal within the known dynamic range
of said assay for said target analyte is used to determine said
presence, absence, activity or concentration of said target
analyte.
34. The method of claim 33, wherein said computer system stores the
cumulative change in said light signal in two or more of said time
intervals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent application
Ser. No. 10/032,790, filed on Oct. 24, 2001, which application is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for increasing the dynamic
range and accuracy of assays, especially binding assays. The
invention further relates to apparatus capable of implementing such
methods.
BACKGROUND OF THE INVENTION
[0003] Biochemical and biological assays are designed to test for
activity in a broad range of systems ranging from protein-protein
interactions, enzyme catalysis, small molecule-protein binding, to
cellular functions.
[0004] In general, fluorescent, chemiluminecent and other assay
formats comprise three distinguishable response ranges. Where the
amount of analyte being assayed is within the dynamic range of the
assay, the reported signal will be dependent upon the amount of
analyte present. Where the amount of analyte exceeds the dynamic
range of the assay, saturation will occur and the reported signal
will not be indicative of the true analyte concentration. Likewise,
where the amount of analyte present in the sample falls below the
threshold of the assay's dynamic range, the assay may be
insufficiently sensitive to the actual analyte concentration, and
the reported signal will also not be indicative of the true analyte
concentration.
[0005] Two approaches have conventionally been employed to address
this problem. In the first, multiple dilutions or concentrations of
a sample are made and then assayed for a defined time period and
the results are evaluated against that of a "standard curve" of
assay results obtained with analyte of varying but known
concentration. In the second approach, an amount of sample is
assayed for multiple times, and results falling within the dynamic
range of the assay are used to calculate the analyte's
concentration (see, for example, U.S. Pat. No. 5,306,468 (Anderson
et al.), U.S. Pat. No. 6,212,291 (Wang et al.)).
[0006] U.S. Pat. Nos. 6,270,695; 6,218,137; 6,139,782; 6,090,571
and 6,045,727 (Akhavan-Tafti, et al.) and U.S. Pat. Nos. 6,045,991;
5,965,736; 6,580,963 and 5,772,926 (Akhavan-Tafti) indicate the
possibility of making multiple exposures in chemiluminescent
assays. The use of multiple exposures in photography is also known
(see, for example, U.S. Pat. No. 6,177,958 (Anderson) and U.S. Pat.
No. 5,754,229 (Elabd)).
[0007] Microtiter or multi-well plates are becoming increasingly
popular in various chemical and biological assays. High-density
format plates, such as 384, 864 and 1536 well plates, are beginning
to displace 96-well plates as the plate of choice. Many of the
assays conducted in multiwell plates employ some type of light
detection from the plate as the reporter for positive or negative
assays results. Such assays include fluorescence assays,
chemiluminescence assays (e.g., luciferase-based assays),
phosphorescence assays, scintillation assays, and the like. In
particular, with the advent of solid phase scintillating materials,
capsules and beads, homogeneous scintillation proximity assays
(SPA) are now being performed with increasing frequency in
multiwell plates.
[0008] Detection of light signals from multiwell plates in the past
has typically been done using plate readers, which generally employ
a photodetector, an array of such photodetectors, photomultiplier
tubes or a photodiode array to quantify the amount of light emitted
from different wells. Such plate readers have been disclosed, for
example, by (U.S. Pat. No. 4,810,096 (Russell, et al.) and (U.S.
Pat. No. 5,198,670 (VanCauter, et al.)). Although plate readers can
detect the total light from each well, they have a number of
limitations. For example, plate readers are typically not capable
of resolving discrete light sources in a single well, so they could
not be used, for example, to differentiate light from different
beads in one well. Further, most plate readers have fewer
photodetectors than there are wells in the plate, so at least some
wells must be read serially, adding to the time required to
complete the assays. This becomes a substantial problem in assays
where the light signal is so low that each well needs to be in the
detection field for an extended period of time (e.g., tens of
minutes). In addition, most currently-available plate readers have
been designed for 96-well plates. Although some can be adapted for,
e.g., 384-well plates, the adaptation does not result in any
significant increase in throughput, since a 384-well plate going
through a modified 96-well reader typically takes four times as
long to read as a 96-well plate.
[0009] Another technique that has been applied to the detection of
light from multiwell plates is imaging. Prior art imaging systems
typically comprise a standard 50-55 mm fl. 4 photographic lens
coupled to a camera. While such systems can be used to image an
entire multiwell plate, and theoretically provide resolution of
discrete light points within individual wells, they have poor
sensitivity, even when coupled to efficient cameras, so that many
assays still require imaging times of tens of minutes or more.
Other assays, such as SPA bead-based assays, cannot be performed at
all due to lack of sensitivity. Further, images acquired with such
systems suffer from vignetting and lateral distortion effects,
making it difficult or impossible to compare signals from center
portions of the plate with signals from lateral wells.
[0010] The demand for increased throughput during primary screening
using less reagent is changing the way of drug discovery. High
throughput screening in 96-well format plates is being replaced by
the use of higher density plates, such as 384 and 1536-well
formats. The analysis of radiometric assays by scintillation
counters is becoming limiting since only 12 wells can be counted at
a time. (Manzella S. M., et al. "A biphasic radiometric assay of
glycogenin using the hydrophobic acceptor
n-dodecyl-beta-D-maltoside," Anal. Biochem. 1994 Feb. 1; 216(2):
383-91) (West B. C., et al. "Neutrophil uptake of vaccinia virus in
vitro," J. Infect. Dis. 1987 October; 156(4): 597-606)
(Boonkitticharoen V., et al. "Radiometric assay of bacterial
growth: analysis of factors determining system performance and
optimization of assay technique," J. Nucl. Med. 1987 February;
28(2): 209-170).
[0011] Charge coupled devices (CCD) use a light-sensitive
integrated circuit to store and display data for an image in such a
way that each pixel (picture element) in the image is converted
into an electrical charge, the intensity of which is related to a
color in the color spectrum. Such devices have found use in
chemical assays and radiologic imaging (see, for example, U.S. Pat.
No. 5,306,468 (Anderson et al.), U.S. Pat. No. 6,212,291 (Wang et
al.).
[0012] A CCD reads the light emitted through the electrode and the
signal is sent to a microprocessor which converts the signal to the
desired readout form. Data obtained (and, optionally, recorded) by
the detection device is typically processed, e.g., by digitizing
the image and storing and analyzing the image on a computer
readable medium. A variety of commercially available peripheral
equipment and software is available for digitizing, storing and
analyzing a signal or image. For example, real-time binding and
dissociation can be monitored visually or by video imaging, such as
with a CCD camera and frame grabber software (U.S. Pat. No.
5,599,668 (Stimpson, et al.; U.S. Pat. No. 5,843,651 (Stimpson, et
al.)).
[0013] CCD cameras are used for many applications in biochemistry
and medicine (Dujardin F. H., et al., "Quantitative assessment of
cortical bone remodelling from routine radiographs of total hip
arthroplasty," Med. Eng. Phys. 1996 September; 18(6): 489-94; Houze
T. A., et al., "Detection of thymidylate synthase gene expression
levels in formalin-fixed paraffin embedded tissue by
semiquantitative, nonradioactive reverse transcriptase polymerase
chain reaction," Tumour Biol. 1997; 18(1): 53-68); Innocenti B., et
al., "Imaging extracellular waves of glutamate during calcium
signaling in cultured astrocytes," J. Neurosci. 2000 Mar. 1; 20(5):
1800-8; Katanec D, et al., "Computer assisted densitometric image
analysis (CADIA) of bone density in periradicular bone defects
healing," Coll Antropol--1998 December; 22 Suppl: 7-13; Nilsson H,
et al., "Laser-induced fluorescence studies of the biodistribution
of carotenoporphyrins in mice," Br. J. Cancer 1997; 76(3): 355-64;
O'Rourke B, et al., "High-speed digital imaging of cytosolic Ca2+
and contraction in single cardiomyocytes," Am J Physiol 1990 July;
259(1 Pt 2):H230-42); Peng Q., et al., "Correlation of distribution
of sulphonated aluminium phthalocyanines with their photodynamic
effect in tumour and skin of mice bearing CaD2 mammary carcinoma,"
Br. J. Cancer 1995 September; 72(3): 565-74; Pope A. J., et al.,
"The detection of phthalocyanine fluorescence in normal rat bladder
wall using sensitive digital imaging microscopy," Br. J. Cancer
1991 November; 64(5): 875-9; Yasui T, et al., "Imaging of
Lactobacillus brevis single cells and microcolonies without a
microscope by an ultrasensitive chemiluminescent enzyme immunoassay
with a photon-counting television camera," Appl. Environ.
Microbiol. 1997 November; 63(11): 4528-33; Zhang J. H., et al.
"Development of a carbon dioxide-capture assay in microtiter plate
for aspartyl-beta-hydroxylase," Anal. Biochem. 1999 Jul. 1; 271(2):
137-42).
[0014] Although methods involving multiple exposure times have been
used to identify the optimum desired exposure time in enzymatic
assays, a need remains for automatable methods that can be used to
process simultaneously not only multiple samples, but also multiple
signal generation foci within each sample, each potentially
reporting differing signals in the assay. The present invention
addresses this need.
SUMMARY OF THE INVENTION
[0015] This invention relates to methods for increasing the dynamic
range and accuracy of assays in which the presence or absence of a
light signal is used to assay for the presence, absence, or
concentration of a target analyte. The invention additionally
relates to the embodiment of such methods in which a change (i.e.,
an evolution or loss) of the light signal in two or more time
intervals is used to assay for the presence, absence, or
concentration of the target analyte. The invention further relates
to apparatus capable of implementing such methods. The invention is
particularly amenable for us in increasing the dynamic range and
accuracy of binding assays.
[0016] In its preferred embodiments, the present invention provides
a method to increase the dynamic range of a binding assay,
utilizing multiple exposure or integration times. In the case of
assays that are quantitated from an image generated by a CCD
camera, the method involves the use of multiple exposures of the
assay, each with a different exposure time. Samples that give weak
signals can be detected and quantitated using a relatively longer
exposure, while samples from the same assay that give strong
signals can be detected and quantitated using a relatively shorter
exposure. The process of taking multiple exposures, and the
analysis of the multiple images, including the selection of the
proper exposure to use to quantitate any one particular sample, is
preferably automated. Since, for those samples that give a midrange
signal, each exposure represents an independent measurement of the
binding assay, this method permits an increase in both the
precision and accuracy of the assay.
[0017] In detail, the invention concerns a method for enhancing the
dynamic range of an assay of the presence, absence, activity or
concentration of two or more target analytes in one or more samples
wherein the presence, absence, activity or concentration of the
target analytes is assayed by the emission or quenching of a light
signal, wherein the method comprises the steps:
[0018] (A) conducting an assay for the presence, absence, activity
or concentration of each of the target analytes in the one or more
samples wherein the assays each cause light signals to be emitted
or quenched;
[0019] (B) employing a computer system comprising a CCD camera
detector to detect the light signals, and to generate data
corresponding to the detected signals; and
[0020] (C) causing the computer system to compare the generated
data using data corresponding to the light signal generated by a
known concentration of the target analyte in a known dynamic range
of the assay and report the presence, absence, activity or
concentration of the target analyte; wherein the computer system
causes the CCD camera detector to independently detect sufficient
light signal for each of the target analytes to ensure that the
reported presence, absence, activity or concentration of each
target analyte is determined using data corresponding to a light
signal that is within the known dynamic range of the assay for that
target analyte.
[0021] The invention additionally concerns the embodiment of such
method wherein, for at least one of the target analytes, the
computer system causes the CCD camera detector to detect light
signal cumulatively until a total detected light signal is obtained
that is within the known dynamic range of the assay for the target
analyte; and wherein the total detected light signal is used to
determine the presence, absence, activity or concentration of the
target analyte, as well as the embodiment of such method wherein,
for at least one of the target analytes, the computer system causes
the CCD camera detector to detect light signal discontinuously at
more than one time interval so that a detected light signal is
obtained that is within the known dynamic range of the assay for
the target analyte; and wherein the detected light signal that is
within the known dynamic range of the assay for the target analyte
is used to determine the presence, absence, activity or
concentration of the target analyte.
[0022] The invention additionally concerns the embodiments of such
methods wherein the computer system stores the cumulative change in
the light signal in two or more of the time intervals. The
invention additionally concerns the embodiments of such methods
wherein the method assays the presence, absence, activity or
concentration of more than one target analyte in a sample, and
particularly wherein the method simultaneously or sequentially
assays the presence, absence, activity or concentration of the more
than one target analyte in a sample. The invention additionally
concerns the embodiments of such methods wherein the step (D) is
performed simultaneously or sequentially for each target analyte
being assayed.
[0023] The invention further concerns the embodiments of such
methods wherein the target analytes are enzymes or other proteins
whose expression is characteristic of disease (e.g., bone specific
alkaline phosphatase, aldose reductase, myoglobin, troponin I,
etc.); or co-factors (including vitamins, such as vitamin B12,
folate, T.sub.3, T.sub.4, TU, FT.sub.3, FT.sub.4, etc.), drugs or
metabolites (including anti-cancer drugs, chemotherapeutic drugs,
anti-viral drugs, non-steroidal anti-inflammatory drugs (NSAID),
steroidal anti-inflammatory drugs, anti-fungal drugs, detoxifying
drugs, analgesics, bronchodilators, anti-bacterial drugs,
antibiotic drugs, diuretics, digoxin, anti-metabolites, calcium
channel blockers, drugs for treatment of psoriasis, substances of
abuse (e.g., cocaine, opiates, and other narcotics), pesticides,
herbicides, etc.), cell-surface receptors (including
protein-tyrosine kinase receptors (e.g., EGFR, PDGFR, MCSFR, SCFR,
insulin-R, VEGFR, Trk, Met, Ron, Axl, Eph); or cell-surface
receptors (e.g., receptors for TNF and related factors (e.g., Trk,
Met, Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30, CD27,4-1BB,
LNGFR, OX40), serine-threonine kinase receptors (e.g., TGF.beta.R),
transmembrane 7 or G protein-coupled receptor families (e.g., CCR1,
CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4,
BLR1, BLR2, V28, and class I and class II cytokines), receptors
such as CD4, class I (hematopoietic cytokine) receptors (e.g.,
IL-1.beta., IL-2R .beta. and .gamma. chains, IL-3R.alpha.,
IL-5R.alpha., GMCSFR.alpha., the IL-3/IL-5/GM-CSF receptor common
.beta.-chain, IL-4R.alpha., IL-7R.alpha., IL-9R.alpha., IL-10R,
IL-11R.alpha., IL-13R.alpha., LIFR .beta., TPOR, OBR, IL-6R.alpha.,
gp130, OSMR.beta., GCSFR, IL-11R.alpha., IL-12Rb1 and IL-12Rb2,
GHR, PRL, and EPO), EGFR, PDGFR, MCSFR, SCFR, insulin-R, VEGFR, and
class II receptors (e.g., IFNgR.alpha., IFNgR.beta., IL-10R, tissue
factor receptor (TFR), and IFN.alpha.R1), etc.); or hormones (such
as adrenaline (epinephrine), adrenocorticotropic hormone (ACTH),
androgens (e.g., testosterone), angiotensinogen, antidiuretic
hormone (ADH) (vasopressin), atrial-natriuretic peptide (ANP),
calciferol (vitamin D3), calcitonin, calcitriol, cholecystokinin,
chorionic gonadotropin (CG), dopamine, erythropoietin, estrogens
(e.g., estradiol), follicle-stimulating hormone (FSH), gastrin,
glucagon, glucocorticoids (e.g., cortisol and urinary cortisol),
gonadotropin-releasing hormone (GnRH), gorticotropin-releasing
hormone (CRH), growth hormone (GH), growth hormone-releasing
hormone (GHRH), insulin, insulin-like growth factor-1 (IGF-1),
leptin, luteinizing hormone (LH), melatonin, mineralocorticoids
(e.g., aldosterone), neuropeptide Y, noradrenaline
(norepinephrine), oxytocin, parathyroid hormone (PTH),
progesterone, prolactin (PRL), renin, secretin, somatostatin,
theophylline, thiiodothyronine T3, thrombopoietin,
thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone
(TRH), thyroxine (T4); or cytokines (such as the interleukins
(e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,
IL-13) or TNF.alpha., VEGF, GMCSF, IL-1.beta., FGF.beta.,
INF.gamma., EGF, PDGF, MCSF, SCF, insulin, VEGF, Trk, Met, Ron,
Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB, LNGFR, OX40, TGF.beta.R, or
a ligand of CCR1, CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1,
CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28 receptor, or a receptor of
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13;
or antigens (such as those characteristic of Chlamydia,
Streptococcus pyogenes Group A bacteria, H. pylori, or M.
tuberculosis hepatitis virus, rubella, CMV or immunodeficiency
virus (HIV, FIV), prostate specific antigen, etc.); or antibodies
to such antigens, or autoimmune immunoglobulins, thyroglobulin,
anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins, tumor markers
(e.g., prostate specific antigen, AFP CEA, etc.).
[0024] The invention further concerns the embodiments of such
methods wherein the light signal or the change in light signal is
an evolution or loss of a fluorescent light signal, a
chemiluminescent light signal, an ultraviolet light signal, or a
visible wavelength light signal.
[0025] The invention further concerns an apparatus for enhancing
the dynamic range of an assay of the presence, absence, activity or
concentration of two or more target analytes in one or more
samples, wherein the presence, absence, activity or concentration
of the target analytes is assayed by the emission or quenching of a
light signal, the apparatus comprising:
[0026] (A) one or more containers for receiving a portion of the
one or more samples, the containers additionally containing assay
reagents comprising a compound that, in response to the presence of
a target analyte causes a detectable light signal; and
[0027] (B) a computer system comprising a CCD camera detector, the
computer system being specially adapted to detect the light signal
and generate data corresponding to the detected signal; the
computer system additionally processing a capability for comparing
the generated data with data corresponding to the light signal
generated by a known concentration of the target analyte in a known
dynamic range of the assay and report the presence, absence,
activity or concentration of the target analyte; wherein the
computer system causes the CCD camera detector to independently
detect sufficient light signal for each of the target analytes to
ensure that the reported presence, absence, activity or
concentration of each target analyte is determined using data
corresponding to a light signal that is within the known dynamic
range of the assay for that target analyte.
[0028] The invention additionally concerns the embodiment of such
apparatus wherein, for at least one of the target analytes, the
computer system causes the CCD camera detector to detect light
signal cumulatively until a total detected light signal is obtained
that is within the known dynamic range of the assay for the target
analyte; and wherein the total detected light signal is used to
determine the presence, absence, activity or concentration of the
target analyte, as well as the embodiment of such apparatus
wherein, for at least one of the target analytes, the computer
system causes the CCD camera detector to detect light signal
discontinuously at more than one time interval until a detected
light signal is obtained that is within the known dynamic range of
the assay for the target analyte; and wherein the detected light
signal that is within the known dynamic range of the assay for the
target analyte is used to determine the presence, absence, activity
or concentration of the target analyte.
[0029] The invention additionally concerns the embodiment of such
apparatus wherein the computer system stores the cumulative change
in the light signal in two or more of the time intervals.
[0030] The invention additionally concerns the embodiment of such
apparatus wherein the apparatus assays the presence, absence,
activity or concentration of more than one target analyte in the
same sample, especially wherein the apparatus simultaneously or
sequentially assays the presence, absence, activity or
concentration of the more than one target analyte in the same
sample.
[0031] The invention additionally concerns the embodiment of such
apparatus wherein the step (D) is performed simultaneously or
sequentially for each target analyte being assayed.
[0032] The invention additionally concerns the embodiment of such
apparatus wherein the one or more containers is a multi-well
microtiter plate.
[0033] The invention additionally concerns the embodiment of such
apparatus wherein the target analyte has an activity and wherein
the computer system additionally calculates the rate of a target
analyte activity in the sample.
[0034] The invention additionally concerns the embodiment of such
apparatus wherein the target analyte is selected from the group
consisting of an enzyme, a co-factor, a receptor, a receptor
ligand, a hormone, a cytokine, a blood factor, a virus, an antigen,
a steroid, a drug, and an antibody.
[0035] The invention further concerns the embodiments of such
apparatus wherein the light signal or the change in light signal is
an evolution or quenching (loss) of a fluorescent,
chemiluminescent, ultraviolet, or visible wavelength light
signal.
[0036] The invention particularly concerns the embodiments of such
apparatus wherein multiple analytes are assayed in a single
container (either simultaneously or sequentially) as well as
wherein a single analyte is assayed in a single container.
[0037] The invention further concerns the embodiments of such
apparatus wherein the target analytes are enzymes or other proteins
whose expression is characteristic of disease (e.g., bone specific
alkaline phosphatase, aldose reductase, myoglobin, troponin I,
etc.); or co-factors (including vitamins, such as vitamin B12,
folate, T.sub.3, T.sub.4, TU, FT.sub.3, FT.sub.4, etc.), drugs or
metabolites (including anti-cancer drugs, chemotherapeutic drugs,
anti-viral drugs, non-steroidal anti-inflammatory drugs (NSAID),
steroidal anti-inflammatory drugs, anti-fungal drugs, detoxifying
drugs, analgesics, bronchodilators, anti-bacterial drugs,
antibiotic drugs, diuretics, digoxin, anti-metabolites, calcium
channel blockers, drugs for treatment of psoriasis, substances of
abuse (e.g., cocaine, opiates, and other narcotics), pesticides,
herbicides, etc.), cell-surface receptors (including
protein-tyrosine kinase receptors (e.g., EGFR, PDGFR, MCSFR, SCFR,
insulin-R, VEGFR, Trk, Met, Ron, Axl, Eph); or cell-surface
receptors (e.g., receptors for TNF and related factors (e.g., Trk,
Met, Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30, CD27, 4-1BB,
LNGFR, OX40), serine-threonine kinase receptors (e.g., TGF.beta.R),
transmembrane 7 or G protein-coupled receptor families (e.g., CCR1,
CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4,
BLR1, BLR2, V28, and class I and class II cytokines), receptors
such as CD4, class I (hematopoietic cytokine) receptors (e.g.,
IL-1.beta., IL-2R .beta. and .gamma. chains, IL-3R.alpha.,
IL-5R.alpha., GMCSFR.alpha., the IL-3/IL-5/GM-CSF receptor common
.beta.-chain, IL-4R.alpha., IL-7R.alpha., IL-9R.alpha., IL-10R,
IL-11R.alpha., IL-13R.alpha., LIFR .beta., TPOR, OBR, IL-6R.alpha.,
gp130, OSMR.beta., GCSFR, IL-11R.alpha., IL-12Rb1 and IL-12Rb2,
GHR, PRL, and EPO), EGFR, PDGFR, MCSFR, SCFR, insulin-R, VEGFR, and
class II receptors (e.g., IFNgR.alpha., IFNgR.beta., IL-10R, tissue
factor receptor (TFR), and IFN.alpha.R1), etc.); or hormones (such
as adrenaline (epinephrine), adrenocorticotropic hormone (ACTH),
androgens (e.g., testosterone), angiotensinogen, antidiuretic
hormone (ADH) (vasopressin), atrial-natriuretic peptide (ANP),
calciferol (vitamin D3), calcitonin, calcitriol, cholecystokinin,
chorionic gonadotropin (CG), dopamine, erythropoietin, estrogens
(e.g., estradiol), follicle-stimulating hormone (FSH), gastrin,
glucagon, glucocorticoids (e.g., cortisol and urinary cortisol),
gonadotropin-releasing hormone (GnRH), gorticotropin-releasing
hormone (CRH), growth hormone (GH), growth hormone-releasing
hormone (GHRH), insulin, insulin-like growth factor-1 (IGF-1),
leptin, luteinizing hormone (LH), melatonin, mineralocorticoids
(e.g., aldosterone), neuropeptide Y, noradrenaline
(norepinephrine), oxytocin, parathyroid hormone (PTH),
progesterone, prolactin (PRL), renin, secretin, somatostatin,
theophylline, thiiodothyronine T3, thrombopoietin,
thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone
(TRH), thyroxine (T4); or cytokines (such as the interleukins
(e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,
IL-13) or TNF.alpha., VEGF, GMCSF, IL-1.beta., FGF.beta.,
INF.gamma., EGF, PDGF, MCSF, SCF, insulin, VEGF, Trk, Met, Ron,
Axl, Eph, Fas, CD40, CD30, CD27, 4-1 BB, LNGFR, OX40, TGF.beta.R,
or a ligand of CCR1, CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1,
CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28 receptor, or a receptor of
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13;
or antigens (such as those characteristic of Chlamydia,
Streptococcus pyogenes Group A bacteria, H. pylori, or M.
tuberculosi, hepatitis virus, rubella, CMV or immunodeficiency
virus (HIV, FIV), prostate specific antigen, etc.); or antibodies
to such antigens, or autoimmune immunoglobulins, thyroglobulin,
anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins, tumor markers
(e.g., prostate specific antigen, AFP CEA, etc.).
[0038] The invention further concerns the embodiments of such
apparatus wherein the light signal or the change in light signal is
an evolution or loss of a fluorescent light signal, a
chemiluminescent light signal, an ultraviolet light signal, or a
visible wavelength light signal.
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 shows a depiction of a 2-second exposure (A) and a
0.2 second exposure (B), of the fluorescence obtained in a sandwich
fluorescence assay for interleukin-8.
[0040] FIG. 2 shows the quantified values for the assay data of
FIG. 1 in the range 0-1000 pg/ml.
[0041] FIG. 3 shows the quantified values for the assay data of
FIG. 1 in the range 0-100 pg/ml.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] This invention relates to methods for increasing the dynamic
range and accuracy of assays in which a light signal is used to
assay for the presence, absence, or concentration of a target
analyte. The invention further relates to apparatus capable of
implementing such methods. The invention is particularly amenable
for us in increasing the dynamic range and accuracy of binding
assays.
[0043] As used herein, the term "dynamic range" is intended to
denote the range of target analyte concentration in which the
detected light signal (or a change of the light signal) is
dependent upon the concentration of the target analyte.
[0044] As used herein, the term "target analyte" is intended to
denote a compound or compounds whose presence, absence or
concentration are the object of the assay. Virtually any compound
can be employed as a target analyte in the presence invention.
Without limitation, such analytes may be enzymes, co-factors,
receptors, receptor ligands, hormones, cytokines, blood factors,
viruses, antigens, steroids, drugs, antibodies, etc. For example,
the target analytes of the present invention may include:
[0045] enzymes or other proteins whose expression is characteristic
of disease (e.g., bone specific alkaline phosphatase, aldose
reductase, myoglobin, troponin I, etc.);
[0046] drugs or metabolites (e.g., anti-cancer drugs,
chemotherapeutic drugs, anti-viral drugs, non-steroidal
anti-inflammatory drugs (NSAID), steroidal anti-inflammatory drugs,
anti-fungal drugs, detoxifying drugs, analgesics, bronchodilators,
anti-bacterial drugs, antibiotic drugs, diuretics, digoxin,
anti-metabolites, calcium channel blockers, drugs for treatment of
psoriasis, substances of abuse (e.g., cocaine, opiates, and other
narcotics), pesticides, herbicides, etc.);
[0047] co-factors (including vitamins, such as vitamin B12, folate,
T.sub.3, T.sub.4, TU, FT.sub.3, FT.sub.4, etc.);
[0048] cell-surface receptors (e.g., receptors for TNF and related
factors (e.g., Trk, Met, Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40,
CD30, CD27, 4-1BB, LNGFR, OX40), serine-threonine kinase receptors
(e.g., TGF.beta.R), transmembrane 7 or G protein-coupled receptor
families (e.g., CCR1, CCR2.alpha., .beta., CCR3, CCR4, CCR5, CXCR1,
CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28, and class I and class II
cytokines), receptors such as CD4, class I (hematopoietic cytokine)
receptors (e.g., IL-1.beta., IL-2R .beta. and .gamma. chains,
IL-3R.alpha., IL-5R.alpha., GMCSFR.alpha., the IL-3/IL-5/GM-CSF
receptor common .beta.-chain, IL-4R.alpha., IL-7R.alpha.,
IL-9R.alpha., IL-10R, IL-11R.alpha., IL-13R.alpha., LIFR .beta.,
TPOR, OBR, IL-6R.alpha., gp130, OSMR.beta., GCSFR, IL-11R.alpha.,
IL-12Rb1 and IL-12Rb2, GHR, PRL, and EPO), EGFR, PDGFR, MCSFR,
SCFR, insulin-R, VEGFR, and class II receptors (e.g., IFNgR.alpha.,
IFNgR.beta., IL-10R, tissue factor receptor (TFR), and
IFN.alpha.R1), etc.);
[0049] hormones (such as adrenaline (epinephrine),
adrenocorticotropic hormone (ACTH), androgens (e.g., testosterone),
angiotensinogen, antidiuretic hormone (ADH) (vasopressin),
atrial-natriuretic peptide (ANP), calciferol (vitamin D3),
calcitonin, calcitriol, cholecystokinin, chorionic gonadotropin
(CG), dopamine, erythropoietin, estrogens (e.g., estradiol),
follicle-stimulating hormone (FSH), gastrin, glucagon,
glucocorticoids (e.g., cortisol and urinary cortisol),
gonadotropin-releasing hormone (GnRH), gorticotropin-releasing
hormone (CRH), growth hormone (GH), growth hormone-releasing
hormone (GHRH), insulin, insulin-like growth factor-1 (IGF-1),
leptin, luteinizing hormone (LH), melatonin, mineralocorticoids
(e.g., aldosterone), neuropeptide Y, noradrenaline
(norepinephrine), oxytocin, parathyroid hormone (PTH),
progesterone, prolactin (PRL), renin, secretin, somatostatin,
theophylline, thiiodothyronine T3, thrombopoietin,
thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone
(TRH), thyroxine (T4);
[0050] cytokines (such as the interleukins (e.g., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13) or TNF.alpha., VEGF,
GMCSF, IL-1.beta., FGF.beta., INF.gamma.Y, EGF, PDGF, MCSF, SCF,
insulin, VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27,
4-1BB, LNGFR, OX40, TGF.beta.R, or a ligand of CCR1, CCR2.alpha.,
.beta., CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2,
V28 receptor, or a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-10, IL-12, or IL-13;
[0051] antigens (such as those characteristic of Chlamydia,
Streptococcus pyogenes Group A bacteria, H. pylori, or M.
tuberculosi, hepatitis virus, rubella, CMV or immunodeficiency
virus (HIV, FIV), prostate specific antigen, etc.); or
[0052] antibodies to such antigens, or autoimmune immunoglobulins,
thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM
immunoglobulins, tumor markers (e.g., prostate specific antigen,
AFP CEA, etc.).
[0053] The present invention comprises a method to increase the
dynamic range of such binding assays by utilizing multiple exposure
or signal-integration times. In a preferred embodiment, the binding
assays will involve the change of a detectable fluorescent,
chemiluminescent, calorimetric, radiological, nephelometric,
turbidometric, ultraviolet, or other signal in response to the
presence or absence of the analyte that is the target of the
assay.
[0054] In a further embodiment, the presence, absence, or
concentration of a target analyte will be assayed by a change
(i.e., by the evolution or loss) of a light signal in two or more
time intervals. As used herein, the term "change" of a detectable
signal is intended to include both processes resulting in an
increase in signal (for example, as when a fluorescent product is
produced over time as a consequence of the action of a target
enzyme) as well as processes resulting in a decrease in signal (for
example, as when a fluorescent substrate is consumed over time as a
consequence of the action of a target enzyme). In accordance with
the methods of the present invention, the detected light signal may
involve light of the visible, near-UV, or UV wavelengths, and may
be generated by chemiluminescent, fluorescent (including laser
induced fluorescent), colorimetric, radiological, nephelometric,
turbidometric or other mechanism (for example through the use of
analytes and substrates that emit or quench such light signal in
response to the presence, absence or concentration of the target
analyte).
[0055] Any of a wide variety of such analytes and substrates may be
used in accordance with the principles of the present invention in
order to generate such light signal. In one embodiment, such
analytes and substrates may possess a chemiluminescent moiety.
Suitable chemiluminescent moieties include acridinium esters,
ruthenium complexes, metal complexes (e.g., U.S. Pat. No.
6,281,021, U.S. Pat. No. 5,238,108 and U.S. Pat. No. 5,310,687),
oxalate ester-peroxide combination, etc.)
[0056] Alternatively, such analytes and substrates may possess a
calorimetric moiety. Suitable calorimetric moieties include
thiopeptolides, anthroquinone dyes, 2 methoxy 4 (2 nitrovinyl)
phenyl .beta.-2 acetamido 2 deoxy .beta. D glucopyranoside;
ammonium 5 [4 (2 acetamido 2 deoxy .beta. D glucopyranosyloxy) 3
methoxy phenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate
hydrate; 4 {2 [4 (.beta. D glucosyl pyranosyloxy) 3 methoxy
phenyl]vinyl} 1 methylquinolinium iodide, 2 methoxy 4 (2
nitrovinyl) phenyl .beta. D galactopyranoside, 2 {2 [4 (.beta. D
galactopyranosyloxy)3 methoxyphenyl]vinyl} 1 methyl quinolinium
iodide, 2 {2 [4 (.beta. D galactopyranosyloxy)3
methoxyphenyl]vinyl} 3 methyl benzothiazolium iodide, 2 {2 [4
(.beta. D glucopyranosyloxy) 3 methoxyphenyl]vinyl} 1 methyl
quinolinium iodide, 2 {2 [4 (.beta. D glucopyranosyloxy) 3
methoxyphenyl]vinyl} 1 propyl quinolinium iodide, 2 {2 [4 (.beta. D
glucopyranosyloxy) 3 methoxyphenyl]vinyl} 3 methyl benzothiazolium
iodide, ammonium 5 [4 .beta. D glucopyranosyloxy) 3 methoxy
phenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate hydrate, 2
methoxy 4 (2 nitrovinyl) phenyl acetate, 2 methoxy 4 (2 nitrovinyl)
phenyl propionate, 5 [4 propanoyloxy) 3,5 dimethoxy
phenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate, 5 [4
butanoyloxy) 3,5 dimethoxy phenylmethylene] 2 thioxothiazolin 4 one
3 ethanoate, 5 [4 decanoyloxy) 3,5 dimethoxy phenylmethylene] 2
thioxothiazolin 4 one 3 ethanoate, 5 [4 dodecanoyloxy) 3,5
dimethoxy phenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate, 5
[4 tetradecanoyloxy) 3,5 dimethoxy phenylmethylene] 2
thioxothiazolin 4 one 3 ethanoate, Pyridinium 4 {2 [4
(phosphoroyloxy) 3,5 dimethoxyphenyl]vinyl} 1 propyl quinolinium
iodide, Pyridinium 5 (4 phosphoryloxy 3,5 dimethoxy
phenylmethylene) 3 methyl 2 thioxothiazolin 4 one, etc.
[0057] Preferably, however, the detected light will be fluorescent,
and the analytes and substrates will possess a
fluorescence-generating moiety whose fluorescence is dependent upon
the presence, absence or concentration of the target analyte.
Examples of suitable fluorescence-generating moieties include
rhodamine 110; rhodol; coumarin or a fluorescein compound.
Derivatives of rhodamine 110, rhodol, or fluorescein compounds that
have a 4' or 5' protected carbon may likewise be employed.
Preferred examples of such compounds include 4'(5')thiofluorescein,
4'(5')-aminofluorescein, 4'(5')-carboxyfluorescein- ,
4'(5')-chlorofluorescein, 4'(5')-methylfluorescein,
4'(5')-sulfofluorescein, 4'(5')-aminorhodol, 4'(5')-carboxyrhodol,
4'(5')-chlororhodol, 4'(5')-methylrhodol, 4'(5')-sulforhodol;
4'(5')-aminorhodamine 110, 4'(5')-carboxyrhodamine 110,
4'(5')-chlororhodamine 110, 4'(5')-methylrhodamine 110,
4'(5')-sulforhodamine 110 and 4'(5')thiorhodamine 110. "4'(5')"
means that at the 4 or 5' position the hydrogen atom on the carbon
atom is substituted with a specific organic group or groups as
previously listed. A 7-Amino, or sulfonated coumarin derivative may
likewise be employed.
[0058] In a further embodiment, cellprobe reagents may be employed
as the analyte or substrate. In general such cellprobe reagents
contain an "indicator group" and one, two, three, four or even more
"leaving groups." The "indicator group" of the compound is a
chemical moiety selected for its ability to have a first state when
joined to the leaving group, and a second state when the leaving
group is cleaved from the indicator group by the enzyme. The
indicator group is preferably excitable (caused to fluoresce) at a
wavelength about the visible range, for example, at wavelength
between about 450 to 500 nanometers (nm). The indicator group will
usually emit in the range of about 480 to 620 nm, preferably 500 to
600 nm and more preferably 500 to 550 nm. Auto-fluorescence of the
cell is most prevalent below about 500 nm. The indicator group is
preferably derived from fluorescent, colorimetric, bioluminescent
or chemiluminescent compounds. The indicator group is preferably
quenched when joined to the leaving group. The term quenched means
that the indicator group has substantially less fluorescence or
chemiluminescence when joined to the leaving group compared to its
fluorescence or chemiluminescence after the leaving group has been
cleaved. For example, the enzyme glutamyltranspeptidase reacts with
gammaglutamyl amino acid peptide giving gamma glutamic acid;
trypsin cleaves the peptide at the arginine residue;
aminopeptidase-M hydrolyzes the peptide at the aliphatic amino acid
residue; and chymotrypsin cleaves the peptide at the phenylalanine
residue. Suitable fluorogenic indicator compounds include xanthine
compounds. Preferably, the indicator compounds are rhodamine 110;
rhodol; fluorescein; and coumarin, and their derivatives. While,
for convenience, the invention is described below with respect to
fluorescent leaving groups, it will be appreciated that the leaving
groups may alternatively be enzymatic, colorimetric,
bioluminescent, chemiluminescent, paramagnetic, luminescent,
radioactive, etc.
[0059] Each "leaving group" of the compound is a chemical moiety
selected so that it will be cleaved by the enzyme to be analyzed.
For such embodiment, compounds having a molecular weight of less
than about 5,000 are preferred. The leaving group is selected
according to the enzyme that is to be assayed. The leaving group
will preferably have utility for assaying any of a variety of
cellular enzymes, including proteases, caspases, glycosidases,
glucosidases, carbohydrases, phosphodiesterases, phosphatases,
sulfatases, thioesterases, pyrophosphatases, lipases, esterases,
nucleotidases and nucleosidases, as listed above.
[0060] The leaving group is preferably selected from amino acids,
peptides, saccharides, sulfates, phosphates, esters, phosphate
esters, nucleotides, polynucleotides, nucleic acids, pyrimidines,
purines, nucleosides, lipids and mixtures thereof. For example, a
peptide and a lipid leaving group can be separately attached to a
single assay compound such as rhodamine 110. Other leaving groups
suitable for the enzyme to be assayed can be determined empirically
or obtained from the literature. See, for example, Mentlein, R. et
al., H. R., "Influence of Pregnancy on Dipeptidyl Peptidase IV
Activity (CD26 Leukocyte Differentiation Antigen) of Circulating
Lymphocytes", Eur. J. Clin. Chem. Clin. Biochem., 29, 477-480
(1991); Schon, E. et al., Eur. J. Immunol., 17, 1821-1826 (1987);
Ferrer-Lopez, P. et al., "Heparin Inhibits Neutrophil-Induced
Platelet Activation Via Cathepsin", J. Lab Clin. Med. 119(3),
231-239 (1992); and Royer, G. et al., "Immobilized Derivatives of
Leucine Aminopeptidase and Aminopeptidase M.", J. Biol. Chem.
248(5), 1807-1812 (1973). These references are hereby incorporated
by reference in their entirety.
[0061] Examples of such regents include
(Cbz-Phe-Arg-NH).sub.2-rhodamine and
(Cbz-Pro-Arg-NH).sub.2-rhodamine, which have particularly use in
assays for human plasmin and human thrombin, respectively (Leytus,
S. P. et al., "New class of sensitive and selective fluorogenic
substrates for serine proteases," Biochem. J. 215: 253-260
(1983)).
[0062] Derivatives of the tetrapeptides ala-ala-pro-leu and
ala-ala-pro-val (Beckman Coulter) are preferred assay compounds for
assaying the activity of the closely related enzymes leukocyte
elastase and pancreatic elastase (leukocyte elastase is also known
as neutrophil elastase, EC 3.4.21.37; pancreatic elastase is also
known as EC 3.4.21.36) (Stein, R. L. et al. 1987, "Catalysis by
human leukocyte elastase: Mechanistic insights into specificity
requirements," Biochem. 26: 1301-1305; Stein, R. L. et al. 1987,
"Catalysis by human leukocyte elastase: Proton inventory as a
mechanistic probe," Biochem. 26: 1305-1314). Elastases are defined
by their ability to cleave elastin, the matrix protein that gives
tissues the property of elasticity. Human leukocyte elastase is a
serine protease that is a major component of neutrophil granules
and is essential for defense against infection by invading
microorganisms (Bode, W. et al. 1989, "Human leukocyte and porcine
pancreatic elastase: X-ray crystal structures, mechanism, substrate
specificity and mechanism-based inhibitors," Biochem. 28:
1951-1963).
[0063] Aspartic acid-Rho110 (Beckman Coulter) is a preferred assay
compound for assaying the activity of the Ca-dependent enzyme
aminopeptidase A (aspartate aminopeptidase, angiotensinase A, EC
3.4.11.7). Aminopeptidase A is found in both soluble and
membrane-bound forms. Aminopeptidase A is known to cleave the
N-terminal aspartic acid amino acid of angiotensin I or II
(Jackson, E. K. et al., 1995, "Renin and Angiotensin" in Goodman
and Gilman's The Pharmacological Basis of Therapeutics, Ninth
Edition McGraw-Hill, NY). Aminopeptidase A is also identical to
BP-1/6C3 (Wu, Q. et al., 1991. "Aminopeptidase A activity of the
murine B-lymphocyte differentiation antigen BP-1/6C3," Proc. Natl.
Acad. Sci, USA. 88: 676-680), a molecule found on early lineage B
cells but not on mature lymphocytes. BP-1/6C3 may have a role in
the ability to support long-term growth of B cells (Whitlock, C.
A., et al., 1987. "Bone marrow stromal cell lines with
lymphopoietic activity express high levels of a pre-B
neoplasia-associated molecule," Cell 48: 1009-1021.
[0064] The conversion of non-fluorescent dichlorofluorescein
diacetate (DCFH-DA) (Beckman Coulter) to the highly fluorescent
compound 2',7'-dichlorofluorescein (DCF) is a preferred assay
compound for monitoring the oxidative burst in polymorphonuclear
leukocytes and for determining the presence of peroxides formed
through such oxidative bursts (Bass, D. A. et al. "Flow cytometric
studies of oxidative product formation by neutrophils: a graded
response to membrane stimulation." J. Immunol. 130: 1910-1917). The
enzymes responsible for the oxidative burst are rapidly activated
in stimulated neutrophils (Weiss, S. J. 1989, "Tissue destruction
by neutrophils," N. Eng. J. Med. 320: 365-376). DCFH,PMA.Oxidative
Burst contains the compound phorbol myristate acetate (PMA), an
analogue of the cellular signaling molecule diacylglycerol (DAG)
(Alberts, B. et al., Molecular Biology of the Cell, 2nd Edition.
Garland Publishing, Inc. New York, pg 704). Therefore, the presence
of PMA stimulates processes mediated by DAG, including the
oxidative burst. Additionally, resting cells do not have free
peroxides and the production of peroxides is rapidly activated by
many cell stimuli including the presence of the bacteria or other
foreign organisms (Weiss. S. J. 1989, "Tissue destruction by
neutrophils," N. Eng. J. Med. 320: 365-376). The production of
peroxides due to the oxidative burst can by artificially stimulated
by the addition of the compound phorbol myristate acetate (PMA) to
the neutrophils (CellProbe substrate DCFH,PMA.Oxidative Burst).
DCFH.Peroxides can be used to investigate the effect of other
compounds on the oxidative burst including the chemotactic peptide
f-met-leu-phe and the yeast product zymosan.
[0065] Fluorescein diacetate (FDA) (Beckman Coulter) is a preferred
assay compound for assaying the activity of many different
non-specific esterases in human tissues (Coates, P. M. et al.,
1975, "A preliminary genetic interpretation of the esterase
isozymes of human tissues," Ann. Hum. Genet. Lond. 39: 1-20).
Acetate esterase activity measured with -Napthyl acetate has been
used together with other esterase activities to identify leukocyte
cell types and is generally high in normal monocytes and
megakaryocytes and in blast cells of acute myelomonocytic leukemia,
acute monocytic leukemia and acute erythroleukemia. Nelson, D. A.
et al., 1990, "Leukocyte esterases in Hematology," 4th Edition,
Williams, Beutler, Erslev and Lichtman, Eds. McGraw-Hill.
[0066] Fluorescein di-galactopyranoside (Beckman Coulter) is a
preferred assay compound for assaying the activity of the
galactosidase enzymes (.beta.-galactosidase is also known as
lactase, .beta.-D-galactoside galactohydrolase, EC 3.2.1.23;
.alpha.-galactosidase is also known as melibiase,
.alpha.-D-galactoside galactohydrolase, EC 3.2.1.22) (Jongkind, J.
F. et al., 1986, "Detection of acid-b-galactosidase activity in
viable human fibroblasts by flow cytometry," Cytometry 7: 463-466).
Galactosidase enzymes are lysosomal enzymes that cleave terminal
sugar residues from several physiological substrates, including
glycoproteins. Gal galactosidase contains forms of the substrate
that are hydrolyzed by both b-galactosidase and a-galactosidase.
Impaired galactosidase activity leads to accumulation of partially
digested glycoproteins in the lysosomes (Cotran, R. S. et al.,
1994, Robbins Pathologic Basis of Disease, 5th Edition. W. B.
Saunders Co. pages 138-140). The lysosomes become enlarged, and
disrupt normal cell function. The impaired galactosidase activity
may be due to mutations in the galactosidase genes or in the
processing and transport mechanisms of galactosidase to the
lysosomes.
[0067] Glycine-phenylalanine-glycine-alanine-Rho110 (Beckman
Coulter) is a preferred assay compound for assaying the activity of
the collagenase group of proteolytic enzymes in a screen of several
tetrapeptide derivatives. Collagenases are enzymes that digest the
collagens: macromolecules that form highly organized structures in
connective tissue and extracellular matrix. Collagenases and other
members of the matrix metalloproteinase family contribute to
physiological processes such as postpartum involution of the
uterus, wound healing, joint destruction in arthritis, tumor
invasion and periodontitis. The collagenases are Zn+2 dependent
metallo-enzymes that are synthesized in a pro-enzyme inactive form
(Woessner, J F Jr. 1991. Matrix metalloproteinases and their
inhibitors in connective tissue remodeling. FASEB J. 5:
2145-21541). The production of HOCl during the neutrophil oxidative
burst has been postulated as one mechanism for collagenase
activation in vivo.
[0068] The assay compound, fluorescein di-glucuronide (Beckman
Coulter) is hydrolyzed by the lysosomal enzyme b-glucuronidase
(.beta.-glucuronidase is also known as .beta.-D-glucuroniside
glucuronosohydrolase, EC 3.2.1.31). A derivative of
.beta.-glucuronide has been used to measure degranulation in
polymorphonuclear lymphocytes. (PMNs) in a test of the ability of
different non-steroidal anti-inflammatory drugs (NSAIDS) to inhibit
PMN functions (Kankaanranta, H. et al., 1994, "Effects of
non-steroidal anti-inflammatory drugs on polymorphonuclear
leukocyte functions in vitro: focus on fenamates,"
Naunyn-Schmiedeberg's Arch Pharmacol. 350: 685-691). Peripheral
blood T-lymphocytes display higher .beta.-glucuronidase activity
that peripheral blood B-lymphocytes (Crockard, A. et al., 1982,
"Cytochemistry of acid hydrolases in chronic B- and T-cell
leukemias," Am. J. Clin. Pathol. 78: 437-444). Fluorescein
di-glucuronide is a negatively charged compound. To help other
derivatives of sugars pass through cell membranes in assays of
.beta.-glucosidase, a lysomotropic detergent (N-dodecylimidazole)
was used (Kohen, E. et al., 1993, "An in situ study of
beta-glucosidase activity in normal and gaucher fibroblasts with
fluorogenic probes," Cell Biochem. and Function. 11: 167-177).
[0069] Glycine-proline-Rho110 (Beckman Coulter) is a preferred
assay compound for assaying the activity of the serine protease
dipeptidyl peptidase IV (DPP IV; Xaa-Pro-dipeptidyl-aminopeptidase,
Gly-pro naphthylamidase, EC 3.4.14.5). The membrane bound form of
DPP IV is also known as the T-cell activation cell surface marker
CD26 (Fleischer, B., 1994, "CD26: a surface protease involved in
T-cell activation," Immunol. Today. 15: 180-184). The proteolytic
activity of DPP IV may play an essential role in the signaling
function of CD26 (Hegen, M. et al., 1993, "Enzymatic activity of
CD26 (dipeptidylpeptidase IV) is not required for its signalling
function in T cells," Immunobiology. 189: 483-493; Tanaka, T. et
al., 1993, "The costimulatory activity of the CD26 antigen requires
dipeptidyl peptidase IV enzymatic activity," Proc. Natl. Acad. Sci.
USA. 90: 4586-4590). DPP IV cleaves the N-terminal dipeptide from
oligopeptides with sequences analogous to the N-terminal sequence
of signaling molecules IL-1b, IL-2 and TNF-b, but does not have
activity against intact recombinant molecules (Hoffmann, T. et al.
1993, "Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13)
catalyzed hydrolysis of cytokines and peptides with N-terminal
cytokine sequences," FEBS Letters. 336: 61-64). Studies of
dipeptidyl peptidase IV activity with GP DPP IV suggest that DPP IV
is upregulated in mature thymocytes and among thymocytes which are
undergoing programmed cell death (apoptosis) (Ruiz, P. et al.,
1996, "Cytofluorographic evidence thatthymocyte dipeptidyl
peptidase IV (CD26) activity is altered with stage of ontogeny and
apoptotic status," Cytometry. 23: 322-329.
[0070] Glycine-proline-leucine-glycine-proline-Rho110 (Beckman
Coulter) is a preferred assay compound for assaying the activity of
the collagenase group of proteolytic enzymes. Collagenases are Zn+2
dependent metallo-enzymes that are synthesized in a pro-enzyme
inactive form 1. (Collagenases digest the collagens: macromolecules
that form highly organized structures in connective tissue and
extracellular matrix. Collagenases and other members of the matrix
metalloproteinase family contribute to physiological processes such
as postpartum involution of the uterus, wound healing, joint
destruction in arthritis, tumor invasion and periodontitis
(Woessner, J. F. Jr., 1991, "Matrix metalloproteinases and their
inhibitors in connective tissue remodeling," FASEB J. 5:
2145-2154). In a detailed study of the mechanism of hydrolysis of
fluorescent derivatives of GPLGP, Kojima et al. found that a
collagenase-like peptidase cleaved the substrate at the peptide
bond between leu and gly (Kojima, K. et al., 1979, "A new and
highly sensitive fluorescence assay for collagenase-like peptidase
activity," Anal. Biochem. 100: 43-50).
[0071] Lys-Rho110 (Beckman Coulter) is a preferred assay compound
for assaying the activity of aminopeptidase B (EC 3.4.11.6). The
aminopeptidases are a group of enzymes which hydrolyze peptide
bonds near the N-terminus of polypeptides (International Union of
Biochemistry and Molecular Biology. Enzyme Nomenclature. 1992.
Academic Press, San Diego). Aminopeptidase B has been purified from
the cytosolic fraction of human liver and skeletal muscle and shown
to act on synthetic lysyl- or arginyl-substrates. Aminopeptidase B
is activated by Cl-1 or Br-1 ions and inhibited by chelating agents
and bestatin (Sanderink, G. J. et al., 1988, "Human
Aminopeptidases: A Review of the Literature. J. Clin. Chem. Clin.
Biochem. 26: 795-807).
[0072] Fluorescein di-phosphate (Beckman Coulter) is a preferred
assay compound for assaying the activity of the enzyme acid
phosphatase (Acid phosphatase is also known as EC 3.1.3.2) (Rotman,
B. et al., 1963, "Fluorogenic substrates for b-D-galactosidases and
phosphatases derived from fluorescein (3,6-dihydroxyfluoran) and
its monomethyl ether,". Proc. Nat. Acad. Sci. USA 50: 1-6). Assays
of acid phosphatase activity have been used together with assays of
esterase activity to identify many different cell types. Monocytes,
neutrophils and T-lymphocytes have relatively high acid phosphatase
activity while B-lymphocytes have relatively low acid phosphatase
activity. (Crockard, A. et al., 1982, "Cytochemistry of acid
hydrolases in chronic B- and T-cell leukemias," Am. J. Clin.
Pathol. 78: 437-444; Li, C. Y. et al., 1970, "Acid phosphatase
isoenzyme in human leukocytes in normal and pathologic conditions,"
J. Histochem. Cytochem. 18: 473-481). In addition, blast cells of
acute promyelocytic leukemia and acute myelomonocytic leukemia have
been shown to have relatively high acid phosphatase activity
(Nelson, D. A. et al. 1990, "Leukocyte esterases in Hematology
Fourth Edition," Williams W J, Beutler E, Erslev A J and Lichtman M
A eds. McGraw Hill, New York.
[0073] Arginine-Rho 110 (Beckman Coulter) is a preferred assay
compound for assaying the activity of aminopeptidase B (arginyl
aminopeptidase, EC 3.4.11.6). The aminopeptidases are a group of
enzymes which hydrolyze peptide bonds near the N-terminus of
polypeptides (International Union of Biochemistry and Molecular
Biology. Enzyme Nomenclature. 1992. Academic Press, San Diego).
Aminopeptidase B has been purified from the cytosolic fraction of
human liver and skeletal muscle and shown to act on synthetic
lysyl- or arginyl-substrates. Aminopeptidase B is activated by Cl-1
or Br-1 ions and inhibited by chelating agents and bestatin
(Sanderink, G. J. et al., 1988, "Human Aminopeptidases: A Review of
the Literature," J. Clin. Chem. Clin. Biochem. 26: 795-807.
[0074] Arg-Gly-Glu-Ser-Rho110 (Beckman Coulter) is a preferred
assay compound for assaying the activity of the closely related
enzymes leukocyte elastase and pancreatic elastase (leukocyte
elastase: neutrophil elastase, EC 3.4.21.37 pancreatic elastase: EC
3.4.21.36). Leukocyte elastase is a serine protease that is a major
component of neutrophil granules and is essential for phagocytosis
and defense against infection by invading microorganisms (Bode, W.
et al., 1989, "Human leukocyte and porcine pancreatic elastase:
X-ray crystal structures, mechanism, substrate specificity and
mechanism-based inhibitors," Biochem. 28: 1951-1963). The
tetrapeptide RGES is part of the sequence of fibronectin (Gartner,
T. K. et al., 1985, "The tetrapeptide analogue of the alpha chain
and decapeptide analogue of the gamma chain of fibrinogen bind to
different sites on the platelet fibrinogen receptor," Blood. 66
Suppl 1: 305a), which is cleaved by human leukocyte elastase
(McDonald, J. A. et al., 1980, "Degradation of fibronectin by human
leukocyte elastase," J. Biol. Chem. 255: 8848-8858).
[0075] The assay compound, threonine-proline-Rho110 (Beckman
Coulter) was identified as a substrate for cathepsin C
(dipeptidyl-peptidase I, EC 3.4.14.1) and cathepsin G (EC
3.4.21.19) by a screen of many different dipeptide derivatives.
Cathepsin C (DPPI) is a lysosomal cysteine peptidase that is found
in relative abundance in cytotoxic cells (Thiele, D. L. et al.,
1990, "Mechanism of L-leucyl-L-leucine methyl ester-mediated
killing of cytotoxic lymphocytes: Dependence on a lysosomal thiol
protease, dipeptidyl peptidase I, that is enriched in these cells,"
Proc. Natl. Acad. Sci. USA. 87: 83-87). Cathepsin G is a serine
endopeptidase that is a major component of the azurophil granules
of polymorphonuclear leukocytes. Cathepsin G activity is high in
promonocytic cells, but reduced in mature monocytes (Hohn, P. A. et
al., 1989, "Genomic organization and chromosomal localization of
the human cathepsin G gene," J. Biol. Chem. 264: 13412-13419.
[0076] Other suitable leaving groups are described in Table 1 of
U.S. Pat. No. 5,698,411 (Lucas, et al.) and Landrum et al. (U.S.
Pat. No. 5,976,822), and include: (Acetyl-.alpha.-D-glucopyranosyl)
Rho 110; (Adenine).sub.2 Rho 110; (Adenosine Monophosphate).sub.2
Rho 110; (Adenosine) Rho 110; (B-D-Galactopyranoside).sub.2 Rho
110; (B-D-glucuronide).sub.2 Rho 110; (Butyrl-Thiocholine).sub.2;
(Cytosine).sub.2 Rho 110; (Guanine).sub.2 Rho 110; (H Gly).sub.2
Rho 110; (H Gly-Arg).sub.2 Rho 110; (H Gly-Gly-Arg).sub.2 Rho 110;
(H Gly-Leu).sub.2 Rho 110; (H Gly-Phe-Gly-Ala).sub.2 Rho 110; (H
Gly-Pro-Leu-Gly-Pro).sub.2 Rho 110; (H-Gly).sub.2-4'chloro-Rho 110;
(H-Gly).sub.2 Rho 110; (H-Gly-Ala-Ala-Ala).sub.2 Rho 110;
(H-Gly-Arg).sub.2 Rho 110; (H-Gly-Gly-Arg).sub.2 Rho 110;
(H-Gly-Pro).sub.2 Rho 110; (H-Gly-Pro-Leu-Gly-Pro) Rho 110;
(Hippuryl-His-Leu).sub.2 Rho 110; (H-L Ala-Ala-Ala-Ala).sub.2 Rho
110; (H-L Ala-Pro).sub.2 Rho 110; (H-L Leu-Leu-Arg).sub.2 Rho 110;
(H-L Lys-Ala).sub.2 Rho 110; (H-L Lys-Ala).sub.2 Rho
110.Sulfo.4TFA; (H-L Lys-Ala-Lys-Ala).sub.2 Rho 110; (H-L
Pro-Arg).sub.2 Rho 110; (H-L-Ala).sub.2-4'chloro-Rho 110;
(H-L-Ala).sub.2-Rho 110; (H-L-Ala-Ala).sub.2 Rho 110;
(H-L-Ala-Ala-Ala).sub.2 Rho 110; (H-L-Ala-Ala-Pro-Ala).sub.2 Rho
110; (H-L-Ala-Ala-Tyr).sub.2 Rho 110; (H-L-Ala-Arg-Arg).sub.2 Rho
110; (H-L-Ala-Gly).sub.2 Rho 110; (H-L-Ala-Phe-Lys).sub.2 Rho 110;
(H-L-Ala-Pro).sub.2-Rho 110; (H-L-Ala-Pro-Ala).sub.2 Rho 110;
(H-L-Arg).sub.2 Rho 110; (H-L-Arg-Arg).sub.2 Rho 110;
(H-L-Arg-Gly-Glu-Ser).sub.2 Rho 110; (H-L-Asp).sub.2-Rho 110;
(H-L-Cys).sub.2-Rho 110; (H-L-Gln-Ser).sub.2 Rho 110;
(H-L-Glu).sub.2-Rho 110; (H-L-Glu-Cys-Gly).sub.2 Rho 110;
(H-L-Glu-Gly-Arg).sub.2 Rho 110; (H-L-Glu-Gly-Phe).sub.2 Rho 110;
(H-L-Glu-Lys-Lys).sub.2 Rho 110; (H-L-Gly-Arg).sub.2-Rho 110;
(H-L-Leu).sub.2-4'chloro-Rho 110; (H-L-Leu).sub.2 Rho 110;
(H-L-Leu-Gly).sub.2 Rho 110; (H-L-Leu-Gly-Leu-Gly).sub.2 Rho 110;
(H-L-Leu-Leu-Arg).sub.2 Rho 110; (H-L-Lys).sub.2 Rho 110;
(H-L-Lys).sub.2-Rho 110; (H-L-Lys-Ala).sub.2-Rho 110;
(H-L-Lys-Ala).sub.2 Rho 110-Sulfo; (H-L-Lys-Ala-Arg-Val).sub.2 Rho
110; (H-L-Lys-Ala-Arg-Val-Phe).sub.2 Rho 110;
(H-L-Lys-Ala-Lys-Ala).sub.2-Rho 110.6TFA; (H-L-Lys-Pro).sub.2 Rho
110; (H-L-Lys-Pro).sub.2-Rho 110; (H-L-Met).sub.2 Rho 110;
(H-L-Phe-Arg).sub.2 Rho 110; (H-L-Pro).sub.2 Rho 110;
(H-L-Pro).sub.2-Rho 110; (H-L-Pro-Arg).sub.2 Rho 110;
(H-L-Pro-Phe-Arg).sub.2 Rho 110; (H-L-Ser).sub.2 Rho 110;
(H-L-Serine Phosphate).sub.2 Rho 110; (H-L-Threonine
Phosphate).sub.2 Rho 110; (H-L-Thr-Pro).sub.2 Rho 110;
(H-L-thyroxine).sub.2 Rho 110; (H-L-Tyrosine Phosphate).sub.2 Rho
110; (H-L-Val-Leu-Lys).sub.2 Rho 110; (H-L-Val-Lys-Val-Lys).sub.2
Rho 110; (H-L-Val-Pro-Arg).sub.2 Rho 110; (H-L-Val-Ser).sub.2 Rho
110; (H-Pro-Arg).sub.2-Rho 110; (N-Acetyl MET).sub.2 Rho 110;
(N-Acetyl-L-Ala).sub.2 FL; (Phosphatidyl-choline).sub- .2 Rho 110;
(Saturated Hydrocarbon).sub.2 Rho 110; (Thymidine).sub.2 Rho 110;
(Triacetin).sub.2 Rho 110; (Unsaturated Hydrocarbon).sub.2 Rho 110;
(Z-Ala-Ala).sub.2 Rho 110; (Z-Ala-Gly).sub.2 Rho 110;
(Z-Thr-Pro).sub.2 Rho 110; (.gamma.-Glu).sub.2 Rho 110;
FL(Acetyl-Choline).sub.2; FL(butyrate).sub.2;
FL(chloroacetate).sub.2; FL(chlorobutyrate).sub.2;
FL(choline).sub.2; FL(heptanoate).sub.2; FL(hexanoate).sub.2;
FL(palmitate).sub.2; FL(phosphate).sub.2; FL(propionate).sub.2;
FL(valerate).sub.2; Fluorescein (acetate).sub.2; H-L-Leu Rhodol;
H-L-Leu Rhodol; Rho 110 (phosphate).sub.2; Rho 110
(Phosphatidyl-choline).sub.2; Rho 110 (Phosphatidylinositol).sub.2;
and Rho 110 (AMP).sub.2.
[0077] When the leaving group of the assay compound is a salt
complex, it will significantly improve the transmission of the
assay compound into the cell (Lucas, et al. (U.S. Pat. No.
5,698,411) and Landrum et al. (U.S. Pat. No. 5,976,822)). The
selection of an appropriate salt complex requires a consideration
of the compatibility with the cell, solubility in the aqueous
media, and cleavage by the enzyme. Care is required in the
selection of the peptide salt since isoenzymes have been found to
be specific in their recognition of particular salts.
[0078] Leaving groups for saccharidases are preferably prepared by
the synthesis of monosaccharides, oligosaccharides or
polysaccharides comprising between one and about ten sugar residues
of the D-configuration. Examples of useful sugars are
monosaccharides-pentoses; ribose; deoxyribose; hexose: glucose,
dextrose, galactose, oligosaccharides-sucrose, lactose, maltose and
polysaccharides like glycogen and starch. The sugar can be an alpha
or beta configuration containing from 3 to 7 and preferably 5 to 6
carbon atoms. Analogs of these sugars can also be suitable for the
invention. Preferably, the D-configuration of the monosaccharide or
disaccharide is utilized. The monosaccharide or disaccharide can be
natural or synthetic in origin.
[0079] Leaving groups for nucleases, nucleotidases, and
nucleosidases are preferably prepared by the synthesis of nucleic
acids, purines, pyrimidines, pentose sugars (i.e., ribose and
deoxyribose) and phosphate ester. Examples are adenine, guanine,
cytosine, uracil and thymine. Leaving groups for restriction
enzymes would include polynucleotides. The nucleic acids contain a
purine or pyrimidine attached to a pentose sugar at the 1-carbon to
N-9 purine or N-1 pyrimidine. A phosphate ester is attached to the
pentose sugar at the 5' position. Analogs of these building blocks
can also be used.
[0080] Leaving groups for lipases are preferably prepared by the
synthesis of simple lipids, compound lipids or derived lipids.
Simple lipids can be esters of fatty acids, triglycerides,
cholesterol esters and vitamin A and D esters. Compound lipids can
be phospholipids, glycolipids (cerebrosides), sulfolipids,
lipoproteins and lipopolysaccharides. Derived lipids can be
saturated and unsaturated fatty acids and mono or diglycerides.
Analogs of these lipids can also be used. Examples of lipids are:
triglycerides--triolein, fatty acids--linoleic, linolenic and
arachidonic; sterols--testosterone, progesterone, cholesterol;
phospholipids--phosphatidic acid, lecithin, cephalin (phosphatidyl
ethanolamine) sphingomyleins; glycolipids--cerebosides,
gangliosides.
[0081] Leaving groups for esterases are preferably prepared by the
synthesis of carboxylic acids comprising between 2 and 30 carbon
atoms. The carboxylic acids can be saturated or unsaturated. The
carboxylic acid preferably contains 2 to 24 carbons and more
preferably 4 to 24 carbon atoms. Analogs of theses carboxylic acids
can also be used. The carboxylic acids can be natural or synthetic
in origin. Examples are butyric, caproic, palmitic, stearic, oleic,
linoleic and linolenic.
[0082] Leaving groups for phosphatases are preferably prepared by
the synthesis of phosphates, phosphatidic acids, phospholipids and
phosphoproteins. Analogs of these compounds can also be used.
Examples are ATP, ADP, AMP and cyclic AMP (c-AMP).
[0083] Leaving groups for peptidases are preferably prepared by the
synthesis of peptides comprising between one and about ten amino
acid residues of the L-configuration. Typically, it has been found
that the synthesis of peptides having more than about six amino
acids produces a low yield. However, where the yield is acceptable,
peptides of greater length can be employed. The amino acids
preferably contain 2-10 and preferably 2-8 carbon atoms. Analogs of
these amino acids can also be suitable for the invention. If the
amino acids are chiral compounds, then they can be present in the
D- or L-form or also as a racemate. Preferably, the L-configuration
of the amino acid is utilized. The amino acids of the oligopeptide
can be natural and/or of synthetic origin. Amino acids of natural
origin, such as occur in proteins and peptide antibiotics, are
preferred. Synthetic amino acids can also be used, such as
pipecolic acid, cyclohexylalanine, phenylglycine,
.alpha.-aminocyclohexylcarboxylic acid, hexahydrotyrosine,
norleucine, or ethionine.
[0084] Suitable methods for synthesizing, purifying, and preparing
such compounds for use in cell-based assays are described in Lucas,
et al. (U.S. Pat. No. 5,698,411) and Landrum et al. (U.S. Pat. No.
5,976,822), herein incorporated by reference.
[0085] In accordance with the methods of the present invention, the
detectable signal is preferably detected with a charge-coupled
device (CCD) camera or similar detector capable of detecting and
storing images resulting from the detected signal. Suitable CCD
cameras are available from Alpha-Innotech (San Leandro, Calif.),
Stratagene (La Jolla, Calif.), and BioRad (Richmond, Calif.), and
Beckman-Coulter (Fullerton, Calif.). The RavidVue.TM.
(Beckman-Coulter) particle shape and size analyzer may be employed
for this purpose.
[0086] For the automated handling and processing of multiple
samples, the SAGIAN.TM. Automated Assay Optimization.TM. System
(Beckman-Coulter), or the FLUOstar 97.TM. or POLARstar.TM. System
(BMG), adapted to detect and store images with a CCD camera may be
used. The SAGIAN.TM. Automated Assay Optimization.TM. System
employs a Biomek.RTM. 2000 Laboratory Automation Workstation
(Beckman-Coulter) with BioWorks.TM. 3.1 Software (Beckman-Coulter).
Automation of the assay can be accomplished using SAGIAN AAO.TM.
Software (Beckman-Coulter) and a computer with Windows.RTM. NT 4.0
SP3 and Excel 97 (Microsoft Corporation). Flurorescence can be
quantified using ImaGene 4.0 assay quantitation software
(BioDiscovery Inc.). The FLUOstar 97.TM./POLARstar.TM. System is a
fully automated microplate-based fluorescence reader developed to
measure data on a vast array of fluorescence assays. Measuring from
above or below the microplate enables both tissue culture and FIA
applications. The POLARstar can detect definitive receptor binding
results through fluorescence polarization readings with 384-well
microplates.
[0087] Other software (e.g., LEADseeker, etc.) may alternatively be
used to facilitate very rapid analysis of high density formats and
permit the ultra-high throughput screening of a range of biological
assays (Fowler A., et al., "A multi-modality assay platform for
ultra-high throughput screening," Curr. Pharm. Biotechnol. 2000
November; 1(3): 265-81).
[0088] In the case of a binding assay that is quantified by
densitometric analysis of a CCD camera-generated image, the dynamic
range of the assay is limited by the dynamic range of the camera.
For any single exposure, weak signals that are below the limit of
detection of the camera will not be detected, while strong signals
that are above the saturation limit of the camera cannot be
quantified. Although the exposure time can be lengthened to detect
and quantify weaker signals, or shortened to detect and quantify
stronger signals, altering the exposure time does not change the
overall dynamic range of the assay. It merely shifts the range to
either lower or higher values.
[0089] In accordance with the methods of the present invention,
multiple measurements of a binding assay, from multiple images of
the same sample generated using multiple exposure times are made.
The dynamic range of the assay is therefore increased, dependent on
the weakest signal detected on the longest exposure, and on the
strongest signal that is below saturation on the shortest
exposure.
[0090] Unlike other conventional detection schemes, such as film,
radiography or calorimetric spectroscopy, the CCD acquires "frames"
of information by detecting the number of fluorescent,
chemiluminescent, or other detectable events occurring over a
selected period of time. Each frame, or a sequence of frames that
have been added or summed to provide an image, can be filtered
using pulse height analysis techniques to substantially reduce or
eliminate background noise. After the first exposure is acquired
(recording the extent of reaction in a first time interval), the
resulting image can be stored and a additional exposures made in
order to record the extent of reaction in additional time
intervals. Each such image provides quantitative information about
the relative extent of the assay occurring within the time interval
involved. Once such multiple images have been obtained, processing
techniques may be employed to determine quantitatively or
qualitatively the extent of the generated signal.
[0091] In one embodiment, the methods of the present invention can
be employed to determine the extent of signal generated across all
prior time occurring intervals tested. As such, the assay reports
the cumulative generation of signal. This aspect of the present
invention permits one to expand the dynamic range of the assay, and
facilitates the automated processing of multiple samples that may
have widely differing concentrations of analyte.
[0092] In a second embodiment, the methods of the present invention
can be employed to determine and compare the extent of signal
generated in each interval tested, so as to permit a determination
of the rate of reaction, and the reaction order.
[0093] In one embodiment, the CCD will be an area detector CCD, and
will be capable of simultaneously recording all light changes
occurring in an area (for example, a microtiter well or plate). In
an alternative embodiment, a scanning detector CCD may be used.
Such a detector scans areas of the assay (for example, individual
wells of a microtiter plate, etc.) and then sums the detected
changes (see. e.g., Messler P., et al., "Instrumentation for
multiwavelengths excitation imaging," J. Neurosci. Methods 1996
November; 69(2): 137-47).
[0094] Any of a wide variety of assay formats may be used in
accordance with the methods of the present invention. They may be
heterogenous or homogeneous, and they may be sequential or
simultaneous. They may be competitive or non-competitive. U.S. Pat.
Nos. 5,563,036; 5,627,080; 5,633,141; 5,679,525; 5,691,147;
5,698,411; 5,747,352; 5,811,526; 5,851,778 and 5,976,822 illustrate
several different assay formats and applications.
[0095] Heterogeneous immunoassay techniques typically involve the
use of a solid phase material to which the reaction product becomes
bound. The reaction product is separated from excess sample, assay
reagents and other substances by removing the solid phase from the
reaction mixture. One type of solid phase immunoassay that may be
used in accordance with the present invention is a sandwich
immunoassay. In the sandwich assay, the more analyte present in the
sample the greater the amount of label present on the solid phase.
This type of assay format is generally preferred, especially for
the visualization of low analyte concentrations, because the
appearance of label on the solid phase is more readily detected. In
the sandwich assay, an anti-analyte antibody (capture reagent) is
bound to the insoluble solid phase material, as described by
Schuurs et al. U.S. Pat. Nos. 3,791,932 and 4,016,043, and by
Pankratz, et al., U.S. Pat. No. 5,876,935. A second anti-analyte
antibody is labeled with a detectable agent to form an indicator
reagent, e.g., the detectable label can be an enzyme which will
react with an enzyme substrate to form a detectable product. If the
analyte is present in the test sample, then the two antibodies form
an immunocomplex with the analyte (i.e., an
antibody/analyte/antibody sandwich), and the amount of indicator
reagent associated with the solid phase is directly proportional to
the amount of analyte in the test sample. When the enzyme substrate
is added, it reacts with the enzyme component of the indicator
reagent to signal the presence or amount of analyte associated with
the solid phase.
[0096] In order to eliminate the bound-free separation step and
reduce the time and equipment needed for a chemical binding assay,
a homogeneous assay format may be used. In such assays, one
component of the binding pair may still be immobilized, however,
the presence of the second component of the binding pair is
detected without a bound-free separation. Examples of homogeneous,
optical methods are the EMIT method of Syva, Inc. (Sunnyvale,
Calif.), which operates through detection of fluorescence
quenching, the laser nephelometry latex particle agglutination
method of Behringwerke (Marburg, Germany), which operates by
detecting changes in light scatter, the LPIA latex particle
agglutination method of Mitsubishi Chemical Industries, the TDX
fluorescence depolarization method of Abbott Laboratories (Abbott
Park, Ill.), and the fluorescence energy transfer method of Cis Bio
International (Paris, France). Any of such assays may be employed
in accordance with the present invention.
[0097] The binding assay of the present invention may be configured
as a competitive assay. In a competitive assay, the more analyte
present in the test sample the lower the amount of label present on
the solid phase. In a manner similar to the sandwich assay, the
competitive assay can involve an anti-analyte binding agent bound
to the insoluble solid phase, however, a labeled analyte, instead
of a labeled second antibody of the sandwich assay, is used as the
indicator reagent. In the competitive assay, the indicator reagent
competes with the test sample analyte to bind the capture reagent
on the solid phase. The amount of captured indicator reagent is
inversely proportional to the amount of analyte present in the test
sample. Smith (U.S. Pat. No. 4,401,764) describes an alternative
competitive assay format using a mixed binding complex which can
bind analyte or labeled analyte but wherein the analyte and labeled
analyte can not simultaneously bind the complex. Clagett (U.S. Pat.
No. 4,746,631) describes an immunoassay method using a reaction
chamber in which an analyte/ligand/marker conjugate is displaced
from the reaction surface in the presence of test sample analyte
and in which the displaced analyte/ligand/marker conjugate is
immobilized at a second reaction site. The conjugate includes
biotin, bovine serum albumin and synthetic peptides as the ligand
component of the conjugate, and enzymes, chemiluminescent
materials, enzyme inhibitors and radionucleotides as the marker
component of the conjugate. Li (U.S. Pat. No. 4,661,444) describes
a competitive immunoassay using a conjugate of an anti-idiotype
antibody and a second antibody, specific for a detectable label,
wherein the detectable response is inversely related to the
presence of analyte in the sample. Allen (EP 177,191) describes a
binding assay involving a conjugate of a ligand analog and a second
reagent, such as fluorescein, wherein the conjugate competes with
the analyte (ligand) in binding to a labeled binding partner
specific for the ligand, and wherein the resultant labeled
conjugate is then separated from the reaction mixture by means of
solid phase carrying a binding partner for the second reagent. This
binding assay format combines the use of a competitive binding
technique and a reverse sandwich assay configuration, i.e., the
binding of conjugate to the labeled binding member prior to
separating conjugate from the mixture by the binding of the
conjugate to the solid phase. The assay result, however, is
determined as in a conventional competitive assay wherein the
amount of label bound to the solid phase is inversely proportional
to the amount of analyte in the test sample. Chieregatt et al. (GB
Patent No. 2,084,317) describe a similar assay format using an
indirectly labeled binding partner specific for the analyte.
Mochida et al. (U.S. Pat. No. 4,185,084) also describe the use of a
double-antigen conjugate which competes with an antigen analyte for
binding to an immobilized antibody and which is then labeled; this
method also results in the detection of label on a solid phase
wherein the amount of label is inversely proportional to the amount
of analyte in the test sample. Sadeh et al. (U.S. Pat. No.
4,243,749) describe a similar enzyme immunoassay wherein a hapten
conjugate competes with analyte for binding to an antibody
immobilized upon a solid phase. Any of such variant assays may be
used in accordance with the present invention.
[0098] In all such assay formats, at least one of the components of
the assay reagents will be labeled or otherwise detectable by the
evolution or quenching of light. Such component may be the analyte
being assayed, or a substrate, co-factor, binding partner, or
product of a reaction or activity of such analyte, etc.
Radioisotopic-binding assay formats (e.g., a radioimmunoassay,
etc.) employ a radioisotope as such label; the signal being
detectable by the evolution of light in the presence of a
fluorescent or fluorogenic moiety (see, Lucas, et al. (U.S. Pat.
No. 5,698,411) and Landrum et al. (U.S. Pat. No. 5,976,822).
Enzymatic-binding assay formats (e.g., an ELISA, etc.) employ an
enzyme as a label; the signal being detectable by the evolution of
color or light in the presence of a chromogenic or fluorogenic
moiety. Other labels, such as paramagnetic labels, materials used
as colored particles, latex particles, colloidal metals, such as
selenium and gold, and dye particles (see, U.S. Pat. Nos.
4,313,734; 4,373,932, and 5,501,985) may also be employed.
[0099] All publications and patents 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. Having now
generally described the invention, the same will be more readily
understood through reference to the following examples, which are
provided by way of illustration, and are not intended to be
limiting of the present invention, unless specified.
[0100] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
Enhanced Detection Assay of Interleukin-8 (IL-8)
[0101] In order to illustrate the principles of preferred
embodiments of the present invention, a sandwich immunoassay for
interleukin-8 activity was conducted and the fluorescence of
anti-IL8 detection antibodies was recorded with a CCD camera. The
results obtained are shown in FIGS. 1-3.
[0102] Image A in FIG. 1 is a depiction of a 2-second exposure, in
which the 5 pg/ml sample is easily detected, but in which the 100
and 1,000 pg/ml samples are off-scale, having saturated the pixels
of the CCD camera. Image B in FIG. 1 is a depiction of the result
of a shorter (0.2-second) exposure of the same plate. The signal
from the 1,000 and 100 pg/ml samples can be easily detected and
quantified, but the signal from the 5 pg/ml sample is barely
detectable.
[0103] FIGS. 2 and 3 show the quantified values for the assay.
Fluorescence was quantified using ImaGene 4.0 assay quantitation
software (BioDiscovery Inc.). Each point represents the signal mean
from 9 spots; values from the 2-second exposure are shown by a
solid line, values from the 0.2-second exposure are shown by a
dashed line. The same data is plotted in FIGS. 2 and 3, with
differing x-axis scale. FIGS. 2 and 3 show that relatively high
IL-8 analyte concentrations (between approximately 100 and 1000
pg/ml) can be reliably quantified from the 0.2-second exposure
(dashed line, FIG. 2), while relatively low analyte concentrations
(between approximately 5 to 100 pg/ml) can be reliably quantified
from the longer, 2-second exposure (solid line; FIG. 3). Each image
from a multiple-exposure assay is analyzed and quantified in a
manner identical to a single-exposure assay, so that this method
does not add significant complexity to the performance or analysis
of the assay. Furthermore, because the analysis of each image is
identical, the method is easily automated.
[0104] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
* * * * *