U.S. patent application number 17/507320 was filed with the patent office on 2022-04-14 for rapid measurement of total vitamin d in blood.
The applicant listed for this patent is Labrador Diagnostics LLC. Invention is credited to Tina Noyes.
Application Number | 20220113322 17/507320 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-14 |
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United States Patent
Application |
20220113322 |
Kind Code |
A1 |
Noyes; Tina |
April 14, 2022 |
RAPID MEASUREMENT OF TOTAL VITAMIN D IN BLOOD
Abstract
Assays for rapid measurement of total vitamin D in blood are
provided. Vitamin D is measured following the rapid and
irreversible release of vitamin D due to denaturation and digestion
of vitamin D binding proteins by aspartyl peptidases (e.g., pepsin)
under acidic conditions. Such measurements may be made using a
vitamin D binder (e.g., an antibody) to measure competition between
free vitamin D and added, labeled vitamin D. Synergy between
denaturation and degradation is believed to provide more rapid and
more complete release of vitamin D than would occur with acid or
enzyme alone. These measurements may be made using small amounts of
whole blood, serum, or plasma, and are suitable for use in
automated devices. These methods provide the advantages of reduced
cost, increased speed, reduced discomfort to the subject, and
increased availability and ease of use. Reagents, kits, devices,
and systems for these assays are also disclosed.
Inventors: |
Noyes; Tina; (East Palo
Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Labrador Diagnostics LLC |
Healdsburg |
CA |
US |
|
|
Appl. No.: |
17/507320 |
Filed: |
October 21, 2021 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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15996955 |
Jun 4, 2018 |
11156618 |
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17507320 |
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15658920 |
Jul 25, 2017 |
10073103 |
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15996955 |
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14819259 |
Aug 5, 2015 |
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15658920 |
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14203239 |
Mar 10, 2014 |
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14819259 |
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61776641 |
Mar 11, 2013 |
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International
Class: |
G01N 33/82 20060101
G01N033/82 |
Claims
1-61. (canceled)
62. A kit for determining the amount of vitamin D in a blood
sample, the kit comprising: a low pH composition comprising an
aspartyl peptidase active at low pH; a neutral pH buffer; and
instructions for use for performing a method comprising: contacting
said blood sample with said low pH composition comprising aspartyl
peptidase active at low pH, whereby a low pH diluted blood sample
composition is formed, effective to at least partially denature and
at least partially digest a vitamin D binding protein in the blood
sample; contacting said low pH diluted blood sample composition
with said neutral pH buffer, whereby a neutral pH diluted blood
sample composition is formed, effective that said aspartyl
peptidase is inactivated; and measuring the amount of said vitamin
D in said neutral pH diluted blood sample, wherein said measuring
comprises using a competition assay measurement.
63. The kit of claim 62 further comprising a container for holding
the low pH composition or the neutral pH buffer.
64. The kit of claim 63, wherein said container comprises a septum,
barrier, cap, or closure effective to prevent evaporation from the
container.
65. The kit of claim 64, wherein said septum, barrier, cap, or
closure is effective to reduce or prevent loss of the low pH
composition or the neutral pH buffer from said container.
66. The kit of claim 62, wherein said low pH composition further
comprises a buffer.
67. The kit of claim 66, wherein said buffer comprises a buffer
containing a citrate.
68. The kit of claim 62, further comprising a vitamin D binder.
69. The kit of claim 68 wherein said instructions for use further
includes instructions for contacting said neutral pH diluted blood
sample composition with the vitamin D binder, wherein said vitamin
D binder binds specifically to vitamin D, wherein said vitamin D
binder is selected from an antibody, an antibody fragment, and an
aptamer.
70. The kit of claim 62, further comprising labeled vitamin D.
71. The kit of claim 70 wherein said instructions for use further
includes instructions for adding said labeled vitamin D to said
neutral pH diluted blood sample composition comprising vitamin D
binder wherein said labeled vitamin D comprises a label selected
from a dye, an epitope tag, a fluorescent moiety, a luminescent
moiety, a chemiluminescent moiety, an enzymatic label, a magnetic
label, a paramagnetic label, a contrast agent, a nanoparticle, a
radioisotope, biotin, streptavidin, and a quencher.
72. The kit of claim 70, wherein said labeled vitamin D comprises
an alkaline phosphatase substrate or a horseradish peroxidase
substrate.
73. The kit of claim 72 wherein said instructions for use further
includes instructions for adding the alkaline phosphatase substrate
or a peroxidase substrate.
74. The kit of claim 73 further comprising capture elements
configured to bind to anti-vitamin D antibody, said capture
elements are selected from a polynucleotide, a polypeptide, a
peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a
polysaccharide, an antibody, an antibody fragment, an antibody
mimic, an immunoadhesin, a receptor, a ligand, biotin, avidin,
streptavidin, Extravidin, neutravidin, an avidin derivative, an
avidin analog, a metal, and an epitope tag.
75. The kit of claim 73, further comprising capture elements
configured to bind to anti-vitamin D antibody, said capture
elements is avidin, streptavidin, biotin, an avidin derivative, or
an analog thereof.
76. The kit of claim 62 wherein said aspartyl peptidase comprises
porcine gastric pepsin.
77. The kit of claim 62 wherein the buffer is effective to provide
a pH of between about pH 2.5 and about pH 4 after sample
addition.
78. The kit of claim 62 wherein said low pH comprises a pH of about
pH 5 or less.
Description
BACKGROUND
[0001] Measurement of small molecule analytes present in blood
samples is important for understanding the health status of a
subject; however, many small molecules are bound by blood proteins
and other molecules that interfere with such measurements. Enzymes
have been used in attempts to free target analytes from binding
proteins; for example, Bates used pepsin treatment (at pH 1) to
free thyroxine from serum proteins for radioimmunoassay
measurements in serum following centrifugation of an acid-treated
sample (U.S. Pat. No. 3,962,039). However, such harsh treatment of
a sample and the use of radioactive tracers to identify analytes
are problematic.
[0002] Vitamin D is a steroid vitamin essential for normal calcium
homeostasis, and thus important, for example, in bone health.
Vitamin D comes in many forms; e.g., vitamin D1 through vitamin D5.
Two forms of vitamin D are important in humans: vitamin D2
(ergocalciferol) and vitamin D3 (cholecalciferol). In addition to
these forms of vitamin D, hydroxy forms of the vitamin may also be
found circulating in the blood of a human subject; for example,
common hydroxyl forms of vitamin D2 include 25-hydroxy vitamin D2
and 1,25-dihydroxy vitamin D2. Common hydroxyl forms of vitamin D3
include 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3. The
major circulating forms of vitamin D in blood are the 25-hydroxyl
forms of vitamin D (25-hydroxy vitamin D3 and 25-hydroxy vitamin
D2).
[0003] Vitamin D3 is formed in the skin from its precursor
7-dehydrocholesterol after ultraviolet irradiation or is absorbed
from the diet. It is further hydroxylated in the liver to
25-hydroxy vitamin D3 as the first step of its conversion in the
kidney to 1,25-dihydroxyvitamin D3, which is the biologically
active form. 25-hydroxy vitamin D3 is the main circulating form of
vitamin D, but 25-hydroxy vitamin D2 is also found especially in
subjects who take certain vitamin supplements. So it is important
to measure both forms of 25-hydroxyvitamin D when monitoring
vitamin D status and the effect of vitamin D2 supplementation on
vitamin D status. Vitamin-D (in all its isoforms) is bound tightly
to Vitamin-D binding protein (VDBP) in blood. In order to measure
vitamin D levels in blood, it has to be extracted or displaced from
the binding protein. Extraction methods are typically slow and
cumbersome.
[0004] Armbruster et al. (U.S. Pat. No. 7,964,363) discuss a method
for measuring vitamin D levels in a blood sample at neutral pH
using a serine protease with endo- and exoproteolytic activity
(e.g., proteinase K, Enzyme Commission (EC) number EC 3.4.21.64) in
order to digest vitamin D binding proteins in blood plasma or
serum; the serine protease is inactivated by addition of a dilution
buffer, which allows subsequent use of a monoclonal antibody in
determining vitamin D from the serum or plasma sample. However,
dilution will reduce the concentration of the protease, rather than
inactivate the protease, or may require excessive dilution which
also dilutes the sample and the analyte, rendering measurement of
the analyte more difficult.
[0005] A method for measuring blood levels of vitamin D that has
become standard is a radioimmunoassay (RIA) method using a
.sup.125I-labeled vitamin D tracer (Hollis et al., Clinical
Chemistry 39:529-533 (1993)), available commercially as the
DiaSorin RIA33 test using methods and equipment from Diasorin
Corporation (Stillwater, Minn.). This RIA method uses acetonitrile
extraction followed by competitive radioimmunoassay using
.sup.125I-labelled 25-hydroxy vitamin D and an antibody to
25-hydroxy vitamin D. A second antibody is used as precipitating
agent. A different assay, the Diasorin Liaison.RTM. test, which
does not use radioactive tracers was also developed by the Diasorin
Corporation. The Diasorin Liaison.RTM. test is a chemiluminescent
assay in which serum is incubated with antivitamin-D coated
microparticles and isoluminol derivative-conjugated 25-hydroxy
vitamin D before measurement of the chemiluminescent signal. Other
common vitamin D assays include the IDS Gamma-B assay (which also
uses acetonitrile extraction to release vitamin D for detection by
antibodies) (ImmunoDiagnostic Systems (IDS) Ltd., Scottsdale,
Ariz., USA), and the Nichols Advantage assay (which uses a
denaturing agent to separate 25-hydroxy vitamin D from its binding
protein) (Nichols Institute Diagnostics, San Clement, Calif.).
[0006] However, comparisons of various vitamin D assays, including
separate measurements of vitamin D2 and vitamin D3, indicate that
the results obtained by different methodologies, and in some cases,
by different laboratories, do not always agree (Binkley et al., J
Clin Endocrinol Metab 89:3152-3157 (2004); Hollis, Clinical
Chemistry 46(10):1657-1661 (2000); Lensmeyer et al., Clinical
Chemistry 52(6):1120-1126 (2006)). For example, reports have been
published suggesting that the Nichols Advantage assay is unable to
measure samples containing substantial amounts of 25-hydroxy
vitamin D2 reliably (Carter et al., Clinical Chemistry
50(11):2195-2197 (2004); Terry et al., Clinical Chemistry
51(8):1565-1566 (2005)).
[0007] Vitamin-D deficiency is associated with bone disease. In its
extreme form when left untreated in children the deficiency leads
to osteomalacia or rickets, a severe chronic and irreversible
condition. Vitamin D supplements provide benefits in the form of
enhanced bone health and decreased mortality in elderly women
taking such supplements. However, excess vitamin D may cause
toxicity; thus, it is believed that vitamin D levels should be
maintained, for example, between about 25 ng/milliliter to about 75
ng/milliliter of blood serum. Measurements of vitamin D are needed
in order to identify abnormal vitamin D levels in a patient and to
confirm response to therapy and the proper maintenance of normal
levels.
[0008] Thus, it would be beneficial to be able to make rapid
measurements of vitamin D in a sample of blood from a subject.
INCORPORATION BY REFERENCE
[0009] All patents, patent applications, and publications mentioned
herein are hereby incorporated by reference in their
entireties.
SUMMARY
[0010] Applicants disclose assay methods and compositions useful
for such methods suitable for rapid measurement of vitamin D in
blood without the use of radioactive isotopes, and without need for
extremely harsh acidic conditions. As disclosed herein, such assay
methods do not require centrifugation. These methods may be
performed using only small amounts of blood, reducing cost of the
analysis and reducing discomfort to the subject. For example, the
methods disclosed herein may be used to measure vitamin D in blood
obtained from a drop, or two drops, or a few drops of blood
obtained from a finger-stick, or from a small (e.g., less than
about 100 .mu.L, or less than about 50 .mu.L) amount of blood
obtained from a vein or artery of a subject. In embodiments, assay
methods disclosed herein are effective for determining the total
concentration of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in
samples of whole blood, blood plasma, or blood serum, with a
reportable range of about 5 ng/mL to about 150 ng/mL. These methods
are suitable for use in automated devices, further reducing cost
and increasing availability of these tests to subjects and
increasing their ease of use. Reagents, kits, devices, and systems
for these assays are disclosed.
[0011] Applicants disclose vitamin D assays and methods using
acidic assay conditions and aspartyl peptidases for measurement of
vitamin D in blood. Use of aspartyl peptidases under acidic
conditions to denature and digest vitamin-D binding proteins
(VDBPs) and other proteins, allows the rapid and irreversible
release of vitamin D in a sample, effective to allow the detection
of, and quantification of, vitamin D in the sample. The synergy
between denaturation and degradation of VDBP is believed to provide
greater, more rapid, and more complete vitamin D release than would
be possible with either condition alone. This enables the release
of vitamin D to occur in a short time, so that no pre-treatment
period, or only a short pre-treatment period, is needed in the
assays disclosed herein. In embodiments, the aspartyl peptidase is
pepsin. Following denaturation and digestion under acidic
conditions (e.g., about pH 2 to about pH 6), the amount of vitamin
D in the sample is measured. In embodiments, such measurements are
made under neutral conditions. In embodiments, such measurements
comprise binding measurements utilizing labeled vitamin D. For
example, competition between exogenously added, labeled vitamin D
and free vitamin D from a sample may be used to measure the amount
of vitamin D present in a blood sample. Interference by VDBPs is
reduced or prevented by the enzymatic degradation of these
proteins, so that competition assays disclosed herein are more
sensitive and accurate than would be otherwise possible in the
absence of such degradation.
[0012] Any aspartyl peptidase active at low pH is suitable for the
practice of methods disclosed herein. For example, pepsin is a
suitable aspartyl peptidase. In embodiments of methods disclosed
herein, an aspartyl peptidase active at low pH may be selected from
pepsin, cathepsin, renin, chymosin, HIV protease, plasmepsin,
retropepsin, and nepenthesin. In embodiments of the methods
disclosed herein, the aspartyl peptidase active at low pH comprises
pepsin. In embodiments of the methods disclosed herein, a low pH
comprises a pH of about pH 5 or less. In embodiments of the methods
disclosed herein, a low pH may comprise a pH of about pH 3 or less.
In embodiments of the methods disclosed herein, a low pH may
comprise a pH of about pH 2 to about pH 6. In embodiments of the
methods disclosed herein, a low pH may comprise a pH of about pH
2.5 to about pH 5.5. In embodiments of the methods disclosed
herein, a low pH may comprise a pH of about pH 3 to about pH 5. In
embodiments of the methods disclosed herein, a low pH may comprise
a pH of about pH 2.5 to about pH 4.
[0013] Accordingly, Applicants disclose methods for determining the
amount of vitamin D in a blood sample, wherein said blood sample
comprises vitamin D and a vitamin D binding protein, the methods
comprising:
[0014] Contacting said blood sample with a low pH composition
comprising an aspartyl peptidase active at low pH, whereby a low pH
diluted blood sample composition is formed, effective to at least
partially denature and at least partially digest said vitamin D
binding protein; and
[0015] Measuring the amount of said vitamin D in said blood sample.
A blood sample may comprise whole blood, blood plasma, blood serum,
or other form of blood sample. In embodiments of the methods
disclosed herein, measuring the amount of vitamin D in a blood
sample comprises a competition assay measurement. In embodiments,
the low pH composition comprises a buffered low pH composition
comprising an aspartyl peptidase active at low pH and a buffer,
wherein the buffer provides a pH of between about pH 2 and about pH
6, or between about pH 2.5 and about pH 5.5, or between about pH 3
and about pH 5, or between about pH 2.5 and about pH 4. In
embodiments, the low pH composition comprises a buffered low pH
composition comprising an aspartyl peptidase active at low pH and a
citrate buffer, wherein the citrate buffer provides a pH of between
about pH 2 and about pH 6, or between about pH 2.5 and about pH
5.5, or between about pH 3 and about pH 5, or between about pH 2.5
and about pH 4.
[0016] Applicants disclose embodiments of methods for determining
the amount of vitamin D in a blood sample, wherein said blood
sample comprises vitamin D and a vitamin D binding protein, the
methods comprising:
[0017] Contacting said blood sample with a low pH composition
comprising an aspartyl peptidase active at low pH, whereby a low pH
diluted blood sample composition is formed, effective to at least
partially denature and at least partially digest said vitamin D
binding protein;
[0018] Contacting said low pH diluted blood sample composition with
a neutral pH composition comprising a neutral pH buffer, whereby a
neutral pH diluted blood sample composition is formed, effective
that said aspartyl peptidase is inactivated; and
[0019] Measuring the amount of said vitamin D in said blood
sample.
[0020] In embodiments, the low pH composition comprises a buffered
low pH composition comprising an aspartyl peptidase active at low
pH and a buffer, wherein the buffer provides a pH of between about
pH 2 and about pH 6, or between about pH 2.5 and about pH 5.5, or
between about pH 3 and about pH 5, or between about pH 2.5 and
about pH 4. In embodiments, the low pH composition comprises a
buffered low pH composition comprising an aspartyl peptidase active
at low pH and a citrate buffer, wherein the citrate buffer provides
a pH of between about pH 2 and about pH 6, or between about pH 2.5
and about pH 5.5, or between about pH 3 and about pH 5, or between
about pH 2.5 and about pH 4.
[0021] Applicants disclose further methods for determining the
amount of vitamin D in a blood sample, wherein said blood sample
comprises vitamin D and a vitamin D binding protein, the methods
comprising:
[0022] Contacting said blood sample with a low pH composition
comprising an aspartyl peptidase active at low pH, whereby a low pH
diluted blood sample composition is formed, effective to at least
partially denature and at least partially digest said vitamin D
binding protein;
[0023] Contacting said low pH diluted blood sample composition with
a neutral pH composition comprising a neutral pH buffer, whereby a
neutral pH diluted blood sample composition is formed, effective
that said aspartyl peptidase is inactivated;
[0024] Contacting said neutral pH diluted blood sample composition
with a vitamin D binder; and Measuring the amount of said vitamin D
in said blood sample.
[0025] In embodiments, the low pH composition comprises a buffered
low pH composition comprising an aspartyl peptidase active at low
pH and a buffer, wherein the buffer provides a pH of between about
pH 2 and about pH 6, or between about pH 2.5 and about pH 5.5, or
between about pH 3 and about pH 5, or between about pH 2.5 and
about pH 4. In embodiments, the low pH composition comprises a
buffered low pH composition comprising an aspartyl peptidase active
at low pH and a citrate buffer, wherein the citrate buffer provides
a pH of between about pH 2 and about pH 6, or between about pH 2.5
and about pH 5.5, or between about pH 3 and about pH 5, or between
about pH 2.5 and about pH 4.
[0026] In embodiments, a vitamin D binder may be an anti-vitamin D
antibody. In embodiments, more than one type of vitamin D binder
may contact the neutral pH diluted blood sample composition. In
embodiments comprising more than one type of vitamin D binder, a
neutral pH diluted blood sample composition may be contacted with a
plurality of types of vitamin D binders that may comprise one, two
or more types of anti-vitamin D antibodies and/or other compounds
which bind vitamin D.
[0027] Applicants also disclose further embodiments of methods as
discussed above, which comprise further steps, including steps
wherein measuring the amount of said vitamin D in said blood sample
comprises:
[0028] Adding labeled vitamin D to said neutral pH diluted blood
sample composition comprising an anti-vitamin D antibody;
[0029] Contacting said neutral pH diluted blood sample composition
comprising an anti-vitamin D antibody with a capture surface,
wherein said capture surface comprises capture elements configured
to bind to said anti-vitamin D antibody; and
[0030] Detecting said labeled vitamin D, wherein the amount of
detected vitamin D provides a measure of the amount of vitamin D
present in the blood sample.
[0031] Applicants also disclose herein embodiments of methods as
discussed above, and which further comprise a washing step prior to
said step of measuring the amount of vitamin D. Applicants also
disclose herein embodiments of methods as discussed above, and
which further comprise a washing step prior to said step of
detecting labeled vitamin D. In embodiments, such a washing step
may be effective to remove unbound labeled vitamin D.
[0032] In embodiments of the methods disclosed herein, said labeled
vitamin D may comprise a label selected from a dye, an epitope tag,
a fluorescent moiety, a luminescent moiety, a chemiluminescent
moiety, an enzymatic label, a magnetic label, a paramagnetic label,
a contrast agent, a nanoparticle, a radioisotope, biotin,
streptavidin, a quencher, and derivatives thereof. In embodiments,
labeled vitamin D comprises an alkaline phosphatase label or a
horseradish peroxidase label.
[0033] In embodiments of the methods disclosed herein,
determination of the amount of vitamin D present in the blood
sample may comprise use of a competition measurement, e.g., a
measurement made in a competition assay. In embodiments of the
methods disclosed herein, determination of the amount of vitamin D
present in the blood sample comprises comparison of the amount of
labeled vitamin D detected with a standard curve obtained from
control vitamin D competition assays. In embodiments, determination
of the amount of vitamin D present in the blood sample may comprise
use of a radioimmunoassay, may comprise use of chromatography, or
may comprise use of spectrophotometry.
[0034] In embodiments of the methods disclosed herein, capture
elements configured to bind to a vitamin D binder, such as an
anti-vitamin D antibody, may be selected from a polynucleotide, a
polypeptide, a peptide nucleic acid, a locked nucleic acid, an
oligosaccharide, a polysaccharide, an antibody, an antibody
fragment, an antibody mimic, an immunoadhesin, a receptor, a
ligand, biotin, avidin, streptavidin, Extravidin, neutravidin,
other avidin derivative or avidin analog, a metal, an epitope tag,
and any portion of any of these. In embodiments of the methods
disclosed herein, capture elements configured to bind to said
anti-vitamin D antibody comprise avidin, streptavidin, or
biotin.
[0035] Vitamin D may take one or more of several different forms.
The naturally occurring form of 25-hydroxy vitamin D is the D3
form; however some vitamin supplements contain 25-hydroxy vitamin
D2 and therefore detection of the D2 form, as well as the D3 form,
is also useful (e.g., individuals taking vitamin D2 supplements may
be at risk for toxicity if vitamin D2 levels are not monitored). In
embodiments of the methods disclosed herein, vitamin D comprises
one or more of vitamin D2; vitamin D3; 25-hydroxy vitamin D2;
25-hydroxy vitamin D3; 1,25-dihydroxy vitamin D2; and
1,25-dihydroxy vitamin D3. In embodiments of the methods disclosed
herein, and of reagents useful in these methods, more than one type
of vitamin D binder may be provided in a reagent. For example, a
type of vitamin D binder may have a binding affinity for vitamin D2
and may have another binding affinity for vitamin D3, or for
25-hydroxy vitamin D2; or for 25-hydroxy vitamin D3; or for
1,25-dihydroxy vitamin D2; or for 1,25-dihydroxy vitamin D3. Such
binding affinities may be the same, or may differ from each other.
In embodiments, a reagent for use in the methods disclosed herein
may include more than one type of vitamin D binder, each of which
type of vitamin D binder may bind more than one form of vitamin D.
For example, a reagent for use in the methods disclosed herein may
comprise two types of vitamin D binders, each of which binds two
forms of vitamin D. In embodiments of such reagents, the relative
amounts of the types of vitamin D binders may be selected so that
the total (combined) binding affinity of the two types of binders
for one form of vitamin D is equal to, or substantially equal to,
the total (combined) binding affinity of the two types of binders
for the other form of vitamin D. In embodiments, an anti-vitamin D
antibody is a type of vitamin D binder, and a different
anti-vitamin D antibody is a different type of vitamin D
binder.
[0036] For example, where a first type of vitamin D binder binds to
both vitamin D2 and to vitamin D3, having a first vitamin D2
binding affinity and a second vitamin D3 affinity, and where a
second type of vitamin D binder binds to both vitamin D2 and to
vitamin D3, having a second vitamin D2 binding affinity and a
second vitamin D3 affinity, and wherein one or both of (i) said
first vitamin D2 binding affinity differs from said second vitamin
D2 binding affinity, and (ii) said first vitamin D3 binding
affinity differs from said second vitamin D3 binding affinity,
effective that the first type of vitamin D binder and the second
type of vitamin D binder together provide a combined vitamin D2
binding affinity and a combined vitamin D3 binding affinity;
Applicants disclose a reagent comprising a first type of vitamin D
binder at a first concentration and a second type of vitamin D
binder at a second concentration, wherein the ratio of said first
concentration to said second concentration is a ratio effective
that said combined vitamin D2 binding affinity is similar to said
combined vitamin D3 binding affinity. Applicants further disclose
methods, as discussed herein, for determining vitamin D in a sample
of whole blood, blood plasma, or blood serum, using a reagent
comprising the first type of vitamin D binder at a first
concentration and the second type of vitamin D binder at a second
concentration, wherein the ratio of said first concentration to
said second concentration is a ratio effective that said combined
vitamin D2 binding affinity is similar to said combined vitamin D3
binding affinity.
[0037] In embodiments of reagents comprising a first type of
vitamin D binder at a first concentration and a second type of
vitamin D binder at a second concentration, and in embodiments of
methods using such reagents, the ratio of said first concentration
to said second concentration is a ratio effective that said
combined vitamin D2 binding affinity is substantially equal to said
combined vitamin D3 binding affinity.
[0038] In embodiments of the methods and of the reagents disclosed
herein, one or more of the types of vitamin D binders in such
reagents comprise a type of an anti-vitamin D antibody. In
embodiments of the methods and of the reagents disclosed herein,
two or more of the types of vitamin D binders in such reagents
comprise types of anti-vitamin D antibodies.
[0039] In embodiments of the methods disclosed herein, a blood
sample may have a volume of less than about 100 .mu.L, or less than
about 50 or less than about 25 or less than about 10 .mu.L. In
embodiments, a blood sample may be obtained by a finger-stick, and
the amount of blood may be an amount obtainable by a finger-stick.
In embodiments, the blood sample may be obtained by use of a
capillary tube or other sampling device. In embodiments of the
methods disclosed herein, determination of the amount of vitamin D
in a blood sample may be performed in less than about one hour, or,
in embodiments, in less than about 40 minutes, or in less than
about 30 minutes, after contacting said blood sample with a low pH
composition comprising an aspartyl peptidase active at low pH. A
blood sample may be diluted with a diluent prior to, or during, the
course of the analysis of the vitamin D level in the blood
sample.
[0040] The assays disclosed herein may be performed on a device, or
on a system, for processing a sample. The assays disclosed herein
can be readily incorporated into and used in an automated assay
device, and in an automated assay system. For example, systems as
disclosed herein may include a communication assembly for
transmitting or receiving a protocol based on the analyte to be
detected (e.g., vitamin D) or based on other analytes to be
detected by the device or system. In embodiments, an assay protocol
may be changed based on optimal scheduling of a plurality of assays
to be performed by a device, or may be changed based on results
previously obtained from a sample from a subject, or based on
results previously obtained from a different sample from the
subject. In embodiments, a communication assembly may comprise a
channel for communicating information from said device to a
computer, said wherein said channel is selected from a computer
network, a telephone network, a metal communication link, an
optical communication link, and a wireless communication link. In
embodiments, systems as disclosed herein may transmit signals to a
central location, or to an end user, and may include a
communication assembly for transmitting such signals. Systems as
disclosed herein may be configured for updating a protocol as
needed or on a regular basis.
[0041] Accordingly, Applicants disclose devices configured to
measure vitamin D in a sample of blood according to a method
disclosed herein. Devices configured to measure vitamin D in a
sample of blood according to a method disclosed herein may be
configured to determine vitamin D from a sample of blood that
comprises no more than about 100 .mu.L of blood, or no more than
about 50 .mu.L of blood, or, in embodiments, wherein said sample of
blood comprises no more than about 25 .mu.L of blood, or wherein
said sample of blood comprises no more than about 10 .mu.L of
blood. Such devices may be configured to measure vitamin D in a
sample of blood in less than about one hour, or, in embodiments, in
less than about 40 minutes, or in less than about 30 minutes.
[0042] Devices disclosed herein may be configured to perform an
assay for the measurement of vitamin D and also to perform an assay
for the measurement of another analyte in the blood sample. Devices
disclosed herein may be configured to perform an assay for the
measurement of vitamin D and also to perform an assay comprising
the measurement of a morphological characteristic of a blood cell
in the blood sample. Devices disclosed herein may be configured to
perform an assay for the measurement of vitamin D and also to
perform an assay comprising the measurement of another blood
analyte, e.g., another analyte relevant to the management of the
calcium status of a subject. Such devices may be configured wherein
the assays, or the order of performance of assays, that are
performed by said device may be altered by communication with
another device.
[0043] Applicants also disclose systems comprising a device as
disclosed herein. In embodiments, the system comprises a device
that is configured to perform an assay for the measurement of
vitamin D and also to perform an assay for the measurement of
another analyte in the blood sample. In embodiments, the system
comprises a device that is configured to perform an assay for the
measurement of vitamin D and also to perform an assay for the
measurement of a morphological characteristic of a blood cell in
the blood sample. In embodiments of such a system, assays, or the
order of performance of assays, that are performed by said device
may be altered by communication with another device.
[0044] Applicants also disclose reagents for use in a method of
measuring vitamin D disclosed herein, the reagents comprising an
aspartyl peptidase and having a pH of between about pH 2 and about
pH 5. In embodiments, the aspartyl peptidase is selected from
pepsin, cathepsin, renin, chymosin, HIV protease, plasmepsin,
retropepsin, and nepenthesin. In embodiments, the aspartyl
peptidase comprises pepsin, and may comprise porcine gastric
pepsin. In embodiments, the reagent has a pH of about pH 5 or less.
In embodiments, the reagent has a pH of about pH 3 or less. In
embodiments, the reagent comprises a buffered low pH composition
comprising an aspartyl peptidase active at low pH and a buffer,
wherein the buffer provides a pH of between about pH 2 and about pH
6, or between about pH 2.5 and about pH 5.5, or between about pH 3
and about pH 5, or between about pH 2.5 and about pH 4. In
embodiments, the reagent comprises a buffered low pH composition
comprising an aspartyl peptidase active at low pH and a citrate
buffer, wherein the citrate buffer provides a pH of between about
pH 2 and about pH 6, or between about pH 2.5 and about pH 5.5, or
between about pH 3 and about pH 5, or between about pH 2.5 and
about pH 4.
[0045] Applicants disclose reagents for use in a method of
measuring vitamin D, comprising binders of vitamin D. For example,
a vitamin D binder may be an anti-vitamin D antibody. In
embodiments, a vitamin D binder may be any compound, including a
polypeptide compound, or a non-polypeptide compound, which
selectively binds vitamin D. In embodiments, a vitamin D binder may
bind more than one form of vitamin D. In embodiments, a vitamin D
binder that binds more than one form of vitamin D may bind a first
form of vitamin D with a higher affinity than the affinity with
which it binds a second form of vitamin D. In embodiments, a
reagent may comprise a vitamin D binder that binds a first form of
vitamin D about twice as well as it binds a second form of vitamin
D, or may bind a first form of vitamin D about three times as well
as it binds a second form of vitamin D, or may bind a first form of
vitamin D many times as well as it binds a second form of vitamin
D.
[0046] Applicants disclose reagents for use in a method of
measuring vitamin D, comprising a first anti-vitamin D antibody and
a second anti-vitamin D antibody, wherein each of said anti-vitamin
D antibodies bind both vitamin D2 and vitamin D3 with vitamin D2
binding affinities and with vitamin D3 affinities, respectively,
and wherein one or both of: (i) the vitamin D2 binding affinity of
said first vitamin D antibody differs from the vitamin D2 binding
affinity of said second vitamin D antibody, and (ii) the vitamin D3
binding affinity of said first vitamin D antibody differs from the
vitamin D3 binding affinity of said second vitamin D antibody,
effective that said first and said second anti-vitamin D antibodies
provide a combined vitamin D2 binding affinity and a combined
vitamin D3 binding affinity, said reagent comprising the first
anti-vitamin D antibody at a first concentration and the second
anti-vitamin D antibody at a second concentration, wherein the
ratio of said first concentration to said second concentration is a
ratio effective that the combined vitamin D2 binding affinity is
equal to the combined vitamin D3 binding affinity. In embodiments,
the concentration of a first antibody may be about twice the
concentration of a second antibody. In embodiments, the reagent may
comprise about 100 ng/ml of a first antibody and may comprise about
50 ng/ml of a second antibody. In embodiments, the reagent may have
a pH of between about pH 7.5 and about pH 8.5.
[0047] Applicants disclose kits for use in a method of measuring
vitamin D in a sample of blood, comprising a reagent as disclosed
herein, and a container. In embodiments, a container of a kit
disclosed herein may comprise a septum, barrier, cap, or closure
effective to prevent evaporation or aerosolization of the reagent.
Such prevention of evaporation or aerosolization of the reagent may
be effective to prevent reagent loss, e.g., over time, due to
spillage, or for other reasons. Such a septum, barrier, cap, or
closure may be effective to prevent contamination of the reagent or
to prevent contamination of a device in which the container is
disposed. In embodiments, a kit for use in a method of measuring
vitamin D in a sample of blood comprises a reagent as disclosed
herein, and instructions for its use in an assay disclosed
herein.
[0048] Assays, methods, reagents, kits, devices, and systems as
disclosed herein provide advantages over prior assays, methods,
reagents, kits, and systems by allowing the rapid and inexpensive
measurement of vitamin D, including vitamin D sub-fractions in a
single assay. By avoiding the extremely harsh conditions of pH 1,
e.g., using buffered solutions, such as citrate buffered solutions
at about pH 2 to about pH 6, or at about pH 2.5 to about pH 4,
while allowing use of aspartyl peptidases active at low pH,
proteins are denatured and digested without substantial
precipitation, allowing the use of optical detection methods, such
as detection of chemiluminescence, luminescence, fluorescence,
turbidity, absorbance, or other optical methods, and maintaining
compatibility with fluid transfer methods using pipettes or other
microfluidic means, and so obviating the need for centrifugation.
Detection by luminescence, chemiluminescence, fluorescence,
turbidity, absorbance, or other optical methods, allows for
avoiding the use of radioactive isotopes for detection of vitamin
D. Use of buffered low pH solutions, such as citrate-buffered low
pH solutions, provides a consistent final pH following mixing with
different clinical samples, allowing for consistent and reliable
assay results.
[0049] The methods and assays disclosed herein allow for
measurement of vitamin D and one or more other analytes from the
same sample of blood, or from the same portion of a sample of
blood. Providing desired measurements in a single assay simplifies
procedures, reduces likelihood of error, reduces variability of
results, and allows for more rapid and more inexpensive procedures.
Accordingly, the assays, methods, reagents, kits, devices, and
systems disclosed herein provide improvements over the art.
[0050] Applicants disclose assay methods for determining vitamin D
in plasma, or serum, or in whole blood. The assay methods disclosed
herein were found to perform well with samples of whole blood,
plasma and serum. Applicants are not aware of any other vitamin D
test method able to measure vitamin D in whole blood. Accordingly,
the assay methods disclosed in the present application provide
advantages and capabilities not available from prior methods.
[0051] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key features or essential features of the claimed subject matter,
nor is it intended to be used to limit the scope of the claimed
subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 provides a schematic of an embodiment of the sample
pretreatment method. In the figure, "D" represents vitamin D, shown
bound to vitamin D binding protein (VDBP) in the upper left of the
figure, and shown free of VDBP elsewhere in the figure.
[0053] FIG. 2 presents an immunoassay method embodiment which uses
the pretreatment method. In the figure, "D" represents vitamin D,
which may be free vitamin D as noted in the figure, and which is
shown as vitamin D bound to anti-vitamin D antibody and as labeled
vitamin D; and "B" represents biotin (part of a biotin-labeled
anti-sheep antibody).
[0054] FIG. 3 provides a key to the assay reagents as illustrated
in FIGS. 1 and 2.
[0055] FIG. 4 shows vitamin D measurements using an
anti-25-hydroxyvitamin D antibody, showing measured values of
25-hydroxyvitamin D3 (vertical axis) plotted against the nominal
amounts of 25-hydroxyvitamin D3 spiked into the low BSA buffer.
[0056] FIG. 5 shows clinical correlation between the results of the
present method using pepsin treatment and an anti-vitamin D
antibody, compared with the results obtained by use of an IDS EIA
Kit.
[0057] FIG. 6A shows vitamin D measurements using an
anti-25-hydroxyvitamin D antibody (Ab2F4), showing measured values
of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 (vertical axis)
plotted against the nominal amounts of 25-hydroxyvitamin D2 and
25-hydroxyvitamin D3 spiked into the low BSA buffer.
[0058] FIG. 6B shows vitamin D measurements using a mixture of two
anti-25-hydroxyvitamin D antibodies (Ab5H10 and Ab2F4, in a ratio
of 2:1 by weight/mL, respectively), showing measured values of
25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 (vertical axis)
plotted against the nominal amounts of 25-hydroxyvitamin D2 and
25-hydroxyvitamin D3 spiked into the low BSA buffer.
[0059] FIG. 7 shows correlation between vitamin D measurements
according to the methods of Example 3 (using two anti-vitamin D
antibodies) as compared to the results of standard methods for
clinical (serum) samples.
[0060] FIG. 8 shows comparisons between results using the present
methods using pepsin treatment in matched samples (from the same
donors) of plasma and of serum.
[0061] FIG. 9 shows comparisons between results obtained in whole
blood (spiked with known amounts of vitamin D) and results obtained
in plasma separated from whole blood using the present methods.
DETAILED DESCRIPTION
[0062] Applicants disclose assays in which a proteolytic enzyme
active under acidic condition, such as an aspartyl peptidase, is
used to release vitamin-D and to digest vitamin-D binding protein
(VDBP) and other proteins in acid conditions. Such other proteins
include, for example, other proteins (such as albumin) which may
bind vitamin D, DNAases, RNAases, and other proteins. The use of an
apartyl peptidase under acidic conditions effective to at least
partially denature and at least partially digest VDBP enables the
release of vitamin D to occur in a short time, so that no
pre-treatment period, or only a short pre-treatment period, is
needed in the assays disclosed herein. Aspartyl peptidases (E.C.
3.4.23) include pepsin, cathepsin, renin, chymosin, HIV protease,
plasmepsin, retropepsin, and nepenthesin, and are most active at
acidic pH (e.g., about pH 2 to 6, or about pH 3 to 5). Pepsin is an
aspartyl peptidase that is well-suited for this purpose because
pepsin acts only in acid conditions which denature the target
protein rendering it more ready for proteolytic attack. In
embodiments, the aspartyl peptidase is a pepsin selected from
porcine pepsin, bovine pepsin, ovine pepsin, murine pepsin, and
human pepsin. In embodiments, the aspartyl peptidase is a cathepsin
selected from cathepsin A, cathepsin B, cathepsin C, cathepsin D,
cathepsin E, cathepsin F, and cathepsin G. In embodiments, the
aspartyl peptidase is a renin selected from porcine renin, bovine
renin, ovine renin, murine renin, and human renin. In embodiments,
the aspartyl peptidase is a chymosin selected from porcine
chymosin, bovine chymosin, ovine chymosin, murine chymosin, and
human chymosin. In embodiments, the aspartyl peptidase is HIV
protease. In embodiments, the aspartyl peptidase is a plasmepsin
selected from porcine plasmepsin, bovine plasmepsin, ovine
plasmepsin, murine plasmepsin, and human plasmepsin. In
embodiments, the aspartyl peptidase is a nepenthesin selected from
porcine nepenthesin, bovine nepenthesin, ovine nepenthesin, murine
nepenthesin, and human nepenthesin.
[0063] Use of buffered solutions, such as citrate buffered
solutions, stabilizes the pH at a desired low pH, providing stable
conditions for reliable and repeatable assay conditions. Such
stability is not believed to be possible, for example, from
solutions merely containing high concentrations of HCl or other
strong acid, which produced turbid or opaque conditions unsuitable
for optical measurements such as luminescence, chemiluminescence,
fluorescence, turbidity, absorbance or other optical measurements.
Citrate buffer was found to provide better results than acid
addition alone, and allowed milder assay conditions (e.g., about pH
2.5 to about pH 4) than obtained with unbuffered strong acid. Use
of buffered solutions, such as citrate buffer, was found to provide
accurate and reliable assays. The citrate buffer seemed more
effective overall at separating the target from the VDBP, and also
was able to produce a consistent final pH after mixing with
different clinical samples.
[0064] Following inactivation and digestion of VDBP by the aspartyl
peptidase active at low pH, effective to release vitamin D bound to
VDBP, detection and/or measurement of vitamin D may be performed at
neutral pH, e.g., by antibody-mediated techniques. Enzymatic
degradation at acid pH is useful, since acid denaturation alone may
not be sufficient to release the vitamin, and since undigested VDBP
might re-nature and re-bind the vitamin if the sample is restored
to neutral pH without digestion of the VDBP. Further advantages of
the methods disclosed herein include use of acid-active enzymes
such as pepsin or other aspartyl peptidases, which are inactivated
by returning the sample to neutral pH (as may be useful where
vitamin D is measured by an immunoassay) and thereafter cannot
digest assay reagents such as antibodies or enzymes and the
like.
[0065] For example, in embodiments of the methods and assays
disclosed herein, biotin-labeled anti-sheep antibody coated on
UltraAvidin.TM. was used as a capture surface in a competitive
enzyme-linked immunosorbent assasy (ELISA). Sample, (serum, plasma,
or whole blood) was diluted and mixed with pepsin in a low pH
buffer to denature and digest vitamin D binding proteins (VDBPs)
and other interfering proteins. The mixture was then further
diluted using a pH 8.0 buffer to inactivate the pepsin, and
anti-25-hydroxy vitamin D antibodies (typically a mixture of
antibodies targeting both 25-hydroxy vitamin D2 and 25-hydroxy
vitamin D3) were added. Following a short incubation period
(typically 10 minutes), an alkaline-phosphatase-labeled 25-hydroxy
vitamin D3 conjugate was added, and the resulting mixture was
incubated in contact with the capture surface. The capture surface
was then washed and the capture surface contacted with alkaline
phosphatase substrate to produce a chemiluminescent signal. The
chemiluminescent signal was read to determine the amount of
25-hydroxy vitamin D in the sample. A greater amount of total
25-hydroxy vitamin D in the sample resulted in lower binding of the
labeled 25-hydroxy vitamin D3 conjugate to the capture antibody.
Thus, the chemiluminescent signal generated by the assay was
inversely proportional to the concentration of 25-hydroxy vitamin D
in the sample.
[0066] The assays and methods disclosed herein can be readily
incorporated into and used in device for processing a sample, or a
system for processing a sample, which may be an automated assay
device, or may be an automated assay system. Such a device, and
such a system, may be useful for the practice of the methods
disclosed herein. For example, a device may be useful for receiving
a sample. A device may be useful for preparing, or for processing a
sample. A device may be useful for performing an assay on a sample.
A device may be useful for obtaining data from a sample. A device
may be useful for transmitting data obtained from a sample. A
device may be useful for disposing of a sample following processing
or assaying of a sample.
[0067] A device may be part of a system, a component of which may
be a sample processing device. A device may be a sample processing
device. A sample processing device may be configured to facilitate
collection of a sample, prepare a sample for a clinical test, or
effect a chemical reaction with one or more reagents or other
chemical or physical processing, as disclosed herein. A sample
processing device may be configured to obtain data from a sample. A
sample processing device may be configured to transmit data
obtained from a sample. A sample processing device may be
configured to analyze data from a sample. A sample processing
device may be configured to communicate with another device, or a
laboratory, or an individual affiliated with a laboratory, to
analyze data obtained from a sample.
[0068] A sample processing device may be configured to be placed in
or on a subject. A sample processing device may be configured to
accept a sample from a subject, either directly or indirectly. A
sample may be, for example, a blood sample (e.g., a sample obtained
from a fingerstick, or from venipuncture, or an arterial blood
sample), a urine sample, a biopsy sample, a tissue slice, stool
sample, or other biological sample; a water sample, a soil sample,
a food sample, an air sample; or other sample. A blood sample may
comprise, e.g., whole blood, plasma, or serum. A sample processing
device may receive a sample from the subject through a housing of
the device. The sample collection may occur at a sample collection
site, or elsewhere. The sample may be provided to the device at a
sample collection site.
[0069] In some embodiments, a sample processing device may be
configured to accept or hold a cartridge. In some embodiments, a
sample processing device may comprise a cartridge. The cartridge
may be removable from the sample processing device. In some
embodiments, a sample may be provided to the cartridge of the
sample processing device. Alternatively, a sample may be provided
to another portion of a sample processing device. The cartridge
and/or device may comprise a sample collection unit that may be
configured to accept a sample.
[0070] A cartridge may include a sample, and may include reagents
for use in processing or testing a sample, disposables for use in
processing or testing a sample, or other materials. Following
placement of a cartridge on, or insertion of a cartridge into, a
sample processing device, one or more components of the cartridge
may be brought into fluid communication with other components of
the sample processing device. For example, if a sample is collected
at a cartridge, the sample may be transferred to other portions of
the sample processing device. Similarly, if one or more reagents
are provided on a cartridge, the reagents may be transferred to
other portions of the sample processing device, or other components
of the sample processing device may be brought to the reagents. In
some embodiments, the reagents or components of a cartridge may
remain on-board the cartridge. In some embodiments, no fluidics are
included that require tubing or that require maintenance (e.g.,
manual or automated maintenance).
[0071] A sample or reagent may be transferred to a device, such as
a sample processing device. A sample or reagent may be transferred
within a device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway from
cartridge to device. Such transfer of sample or reagent may be
accomplished without providing a continuous fluid pathway within a
device. In embodiments, such transfer of sample or reagent may be
accomplished by a sample handling system (e.g., a pipette); for
example, a sample, reagent, or aliquot thereof may be aspirated
into an open-tipped transfer component, such as a pipette tip,
which may be operably connected to a sample handling system which
transfers the tip, with the sample, reagent, or aliquot thereof
contained within the tip, to a location on or within the sample
processing device. The sample, reagent, or aliquot thereof can be
deposited at a location on or within the sample processing device.
Sample and reagent, or multiple reagents, may be mixed using a
sample handling system in a similar manner. One or more components
of the cartridge may be transferred in an automated fashion to
other portions of the sample processing device, and vice versa.
[0072] A device, such as a sample processing device, may have a
fluid handling system. A fluid handling system may perform, or may
aid in performing, transport, dilution, extraction, aliquotting,
mixing, and other actions with a fluid, such as a sample. In some
embodiments, a fluid handling system may be contained within a
device housing. A fluid handling system may permit the collection,
delivery, processing and/or transport of a fluid, dissolution of
dry reagents, mixing of liquid and/or dry reagents with a liquid,
as well as collection, delivery, processing and/or transport of
non-fluidic components, samples, or materials. The fluid may be a
sample, a reagent, diluent, wash, dye, or any other fluid that may
be used by the device, and may include, but not limited to,
homogenous fluids, different liquids, emulsions, suspensions, and
other fluids. A fluid handling system, including without limitation
a pipette, may also be used to transport vessels (with or without
fluid contained therein) around the device. The fluid handling
system may dispense or aspirate a fluid. The sample may include one
or more particulate or solid matter floating within a fluid.
[0073] In embodiments, a fluid handling system may comprise a
pipette, pipette tip, syringe, capillary, or other component. The
fluid handling system may have portion with an interior surface and
an exterior surface and an open end. The fluid handling system may
comprise a pipette, which may include a pipette body and a pipette
nozzle, and may comprise a pipette tip. A pipette tip may or may
not be removable from a pipette nozzle. In embodiments, a fluid
handling system may use a pipette mated with a pipette tip; a
pipette tip may be disposable. A tip may form a fluid-tight seal
when mated with a pipette. A pipette tip may be used once, twice,
or more times. In embodiments, a fluid handling system may use a
pipette or similar device, with or without a pipette tip, to
aspirate, dispense, mix, transport, or otherwise handle the fluid.
The fluid may be dispensed from the fluid handling system when
desired. The fluid may be contained within a pipette tip prior to
being dispensed, e.g., from an orifice in the pipette tip. In
embodiments, or instances during use, all of the fluid may be
dispensed; in other embodiments, or instances during use, a portion
of the fluid within a tip may be dispensed. A pipette may
selectively aspirate a fluid. The pipette may aspirate a selected
amount of fluid. The pipette may be capable of actuating stirring
mechanisms to mix the fluid within the tip or within a vessel. The
pipette may incorporate tips or vessels creating continuous flow
loops for mixing, including of materials or reagents that are in
non-liquid form. A pipette tip may also facilitate mixture by
metered delivery of multiple fluids simultaneously or in sequence,
such as in 2-part substrate reactions.
[0074] The fluid handling system may include one or more
fluidically isolated or hydraulically independent units. For
example, the fluid handling system may include one, two, or more
pipette tips. The pipette tips may be configured to accept and
confine a fluid. The tips may be fluidically isolated from or
hydraulically independent of one another. The fluid contained
within each tip may be fluidically isolated or hydraulically
independent from one fluids in other tips and from other fluids
within the device. The fluidically isolated or hydraulically
independent units may be movable relative to other portions of the
device and/or one another. The fluidically isolated or
hydraulically independent units may be individually movable. A
fluid handling system may comprise one or more base or support. A
base or support may support one or more pipette or pipette units. A
base or support may connect one or more pipettes of the fluid
handling system to one another.
[0075] A sample processing device may be configured to perform
processing steps or actions on a sample obtained from a subject.
Sample processing may include sample preparation, including, e.g.,
sample dilution, division of a sample into aliquots, extraction,
contact with a reagent, filtration, separation, centrifugation, or
other preparatory or processing action or step. A sample processing
device may be configured to perform one or more sample preparation
action or step on the sample. Optionally, a sample may be prepared
for a chemical reaction and/or physical processing step. A sample
preparation action or step may include one or more of the
following: centrifugation, separation, filtration, dilution,
enriching, purification, precipitation, incubation, pipetting,
transport, chromatography, cell lysis, cytometry, pulverization,
grinding, activation, ultrasonication, micro column processing,
processing with magnetic beads, processing with nanoparticles, or
other sample preparation action or steps. For example, sample
preparation may include one or more step to separate blood into
serum and/or particulate fractions, or to separate any other sample
into various components. Sample preparation may include one or more
step to dilute and/or concentrate a sample, such as a blood sample,
or other biological samples. Sample preparation may include adding
an anti-coagulant or other ingredients to a sample. Sample
preparation may also include purification of a sample. In
embodiments, all sample processing, preparation, or assay actions
or steps are performed by a single device. In embodiments, all
sample processing, preparation, or assay actions or steps are
performed within a housing of a single device. In embodiments, most
sample processing, preparation, or assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, many sample processing,
preparation, or assay actions or steps are performed by a single
device, and may be performed within a housing of a single device.
In embodiments, sample processing, preparation, or assay actions or
steps may be performed by more than one device.
[0076] A sample processing device may be configured to run one or
more assay on a sample, and to obtain data from the sample. An
assay may include one or more physical or chemical treatments, and
may include running one or more chemical or physical reactions. A
sample processing device may be configured to perform one, two or
more assays on a small sample of bodily fluid. One or more chemical
reaction may take place on a sample having a volume, as described
elsewhere herein. For example one or more chemical reaction may
take place in a pill having less than femtoliter volumes. In an
instance, the sample collection unit is configured to receive a
volume of the bodily fluid sample equivalent to a single drop or
less of blood or interstitial fluid. In embodiments, the volume of
a sample may be a small volume, where a small volume may be a
volume that is less than about 1000 .mu.L, or less than about 500
.mu.L, or less than about 250 .mu.L, or less than about 150 .mu.L,
or less than about 100 .mu.L, or less than about 75 .mu.L, or less
than about 50 .mu.L, or less than about 40 .mu.L, or less than
about 20 .mu.L, or less than about 10 .mu.L, or other small volume.
In embodiments, all sample assay actions or steps are performed on
a single sample. In embodiments, all sample assay actions or steps
are performed by a single device. In embodiments, all sample assay
actions or steps are performed within a housing of a single device.
In embodiments, most sample assay actions or steps are performed by
a single device, and may be performed within a housing of a single
device. In embodiments, many sample assay actions or steps are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, sample processing, preparation,
or assay actions or steps may be performed by more than one
device.
[0077] A sample processing device may be configured to perform a
plurality of assays on a sample. In embodiments, a sample
processing device may be configured to perform a plurality of
assays on a single sample. In embodiments, a sample processing
device may be configured to perform a plurality of assays on a
single sample, where the sample is a small sample. For example, a
small sample may have a sample volume that is a small volume of
less than about 1000 .mu.L, or less than about 500 .mu.L, or less
than about 250 .mu.L, or less than about 150 .mu.L, or less than
about 100 .mu.L, or less than about 75 .mu.L, or less than about 50
.mu.L, or less than about 40 .mu.L, or less than about 20 .mu.L, or
less than about 10 .mu.L, or other small volume. A sample
processing device may be capable of performing multiplexed assays
on a single sample. A plurality of assays may be run
simultaneously; may be run sequentially; or some assays may be run
simultaneously while others are run sequentially. One or more
control assays and/or calibrators (e.g., including a configuration
with a control of a calibrator for the assay/tests) can also be
incorporated into the device; control assays and assay on
calibrators may be performed simultaneously with assays performed
on a sample, or may be performed before or after assays performed
on a sample, or any combination thereof. In embodiments, all sample
assay actions or steps are performed by a single device. In
embodiments, all of a plurality of assay actions or steps are
performed within a housing of a single device. In embodiments, most
sample assay actions or steps, of a plurality of assays, are
performed by a single device, and may be performed within a housing
of a single device. In embodiments, many sample assay actions or
steps, of a plurality of assays, are performed by a single device,
and may be performed within a housing of a single device. In
embodiments, sample processing, preparation, or assay actions or
steps may be performed by more than one device.
[0078] In embodiments, all of a plurality of assays may be
performed in a short time period. In embodiments, such a short time
period comprises less than about three hours, or less than about
two hours, or less than about one hour, or less than about 40
minutes, or less than about 30 minutes, or less than about 25
minutes, or less than about 20 minutes, or less than about 15
minutes, or less than about 10 minutes, or less than about 5
minutes, or less than about 4 minutes, or less than about 3
minutes, or less than about 2 minutes, or less than about 1 minute,
or other short time period.
[0079] A sample processing device may be configured to detect one
or more signals relating to the sample. A sample processing device
may be configured to identify one or more properties of the sample.
For instance, the sample processing device may be configured to
detect the presence or concentration of one analyte or a plurality
of analytes or a disease condition in the sample (e.g., in or
through a bodily fluid, secretion, tissue, or other sample).
Alternatively, the sample processing device may be configured to
detect a signal or signals that may be analyzed to detect the
presence or concentration of one or more analytes (which may be
indicative of a disease condition) or a disease condition in the
sample. The signals may be analyzed on board the device, or at
another location. Running a clinical test may or may not include
any analysis or comparison of data collected.
[0080] A chemical reaction or other processing step may be
performed, with or without the sample. Examples of steps, tests, or
assays that may be prepared or run by the device may include, but
are not limited to immunoassay, nucleic acid assay, receptor-based
assay, cytometric assay, colorimetric assay, enzymatic assay,
electrophoretic assay, electrochemical assay, spectroscopic assay,
chromatographic assay, microscopic assay, topographic assay,
calorimetric assay, turbidmetric assay, agglutination assay,
radioisotope assay, viscometric assay, coagulation assay, clotting
time assay, protein synthesis assay, histological assay, culture
assay, osmolarity assay, and/or other types of assays,
centrifugation, separation, filtration, dilution, enriching,
purification, precipitation, pulverization, incubation, pipetting,
transport, cell lysis, or other sample preparation action or steps,
or combinations thereof. Steps, tests, or assays that may be
prepared or run by the device may include imaging, including
microscopy, cytometry, and other techniques preparing or utilizing
images. Steps, tests, or assays that may be prepared or run by the
device may further include an assessment of histology, morphology,
kinematics, dynamics, and/or state of a sample, which may include
such assessment for cells.
[0081] A device may be capable of performing all on-board steps
(e.g., steps or actions performed by a single device) in a short
amount of time. A device may be capable of performing all on-board
steps on a single sample in a short amount of time. For example,
from sample collection from a subject to transmitting data and/or
to analysis may take about 3 hours or less, 2 hours or less, 1 hour
or less, 50 minutes or less, 45 minutes or less, 40 minutes or
less, 30 minutes or less, 20 minutes or less, 15 minutes or less,
10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes
or less, 2 minutes or less, or 1 minute or less. The amount of time
from accepting a sample within the device to transmitting data
and/or to analysis from the device regarding such a sample may
depend on the type or number of steps, tests, or assays performed
on the sample. The amount of time from accepting a sample within
the device to transmitting data and/or to analysis from the device
regarding such a sample may take about 3 hours or less, 2 hours or
less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40
minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes
or less, 10 minutes or less, 5 minutes or less, 4 minutes or less,
3 minutes or less, 2 minutes or less, or 1 minute or less.
[0082] A device may be configured to prepare a sample for disposal,
or to dispose of a sample, such as a biological sample, following
processing or assaying of a sample.
[0083] In embodiments, a sample processing device may be configured
to transmit data obtained from a sample. In embodiments, a sample
processing device may be configured to communicate over a network.
A sample processing device may include a communication module that
may interface with the network. A sample processing device may be
connected to the network via a wired connection or wirelessly. The
network may be a local area network (LAN) or a wide area network
(WAN) such as the Internet. In some embodiments, the network may be
a personal area network. The network may include the cloud. The
sample processing device may be connected to the network without
requiring an intermediary device, or an intermediary device may be
required to connect a sample processing device to a network. A
sample processing device may communicate over a network with
another device, which may be any type of networked device,
including but not limited to a personal computer, server computer,
or laptop computer; personal digital assistants (PDAs) such as a
Windows CE device; phones such as cellular phones, smartphones
(e.g., iPhone, Android, Blackberry, etc.), or location-aware
portable phones (such as GPS); a roaming device, such as a
network-connected roaming device; a wireless device such as a
wireless email device or other device capable of communicating
wireless with a computer network; or any other type of network
device that may communicate possibly over a network and handle
electronic transactions. Such communication may include providing
data to a cloud computing infrastructure or any other type of data
storage infrastructure which may be accessed by other devices.
[0084] A sample processing device may provide data regarding a
sample to, e.g., a health care professional, a health care
professional location, such as a laboratory, or an affiliate
thereof. One or more of a laboratory, health care professional, or
subject may have a network device able to receive or access data
provided by the sample processing device. A sample processing
device may be configured to provide data regarding a sample to a
database. A sample processing device may be configured to provide
data regarding a sample to an electronic medical records system, to
a laboratory information system, to a laboratory automation system,
or other system or software. A sample processing device may provide
data in the form of a report.
[0085] A laboratory, device, or other entity or software may
perform analysis on data regarding a sample in real-time. A
software system may perform chemical analysis and/or pathological
analysis, or these could be distributed amongst combinations of
lab, clinical, and specialty or expert personnel. Analysis may
include qualitative and/or quantitative evaluation of a sample.
Data analysis may include a subsequent qualitative and/or
quantitative evaluation of a sample. Optionally, a report may be
generated based on raw data, pre-processed data, or analyzed data.
Such a report may be prepared so as to maintain confidentiality of
the data obtained from the sample, the identity and other
information regarding the subject from whom a sample was obtained,
analysis of the data, and other confidential information. The
report and/or the data may be transmitted to a health care
professional. Data obtained by a sample processing device, or
analysis of such data, or reports, may be provided to a database,
an electronic medical records system, to a laboratory information
system, to a laboratory automation system, or other system or
software.
[0086] Embodiments of devices and systems for measuring vitamin D
in at least a portion of a blood sample; and embodiments of devices
and systems for measuring vitamin D in at least a portion of a
blood sample, and at least one other biologically relevant
attribute from said blood sample from a subject; and description
and disclosure of examples of reagents, assays, methods, kits,
devices, and systems which may use, or be used with, reagents,
assays, methods, kits, devices, and systems disclosed herein may be
found, for example, in U.S. Pat. Nos. 8,088,593; 8,380,541; U.S.
patent application Ser. No. 13/769,798, filed Feb. 18, 2013; U.S.
patent application Ser. No. 13/769,779, filed Feb. 18, 2013; U.S.
patent application Ser. No. 13/244,947 filed Sep. 26, 2011;
PCT/US2012/57155, filed Sep. 25, 2012; and U.S. application Ser.
No. 13/244,947, filed Sep. 26, 2011, the disclosures of which
patents and patent applications are all hereby incorporated by
reference in their entireties.
Definitions
[0087] It should be understood that as used in the description
herein and throughout the claims that follow, the meaning of "a,"
"an," and "the" includes plural reference unless the context
clearly dictates otherwise. Also, as used in the description herein
and throughout the claims that follow, the meaning of "in" includes
"in" and "on" unless the context clearly dictates otherwise. As
used in the description herein and throughout the claims that
follow, the meanings of "and" and "or" include both the conjunctive
and disjunctive and may be used interchangeably unless the context
expressly dictates otherwise. Thus, in contexts where the terms
"and" or "or" are used, usage of such conjunctions do not exclude
an "and/or" meaning unless the context expressly dictates
otherwise.
[0088] As used herein, the term "about" refers to an amount that is
within about 10% of the amount to which the modifier "about"
refers. Thus, for example, the phrase "no more than about 50 .mu.L
of blood" refers to an amount of blood that may be no more than 55
.mu.L in volume.
[0089] As used herein, the term "similar" when applied to numerical
values means that two or more numerical values are close in value
to each other, e.g., a first value differs from a second value by
no more than about 50% of the first value, or by no more than about
40%, or by no more than about 30%, or by no more than about 20%, or
by no more than about 15%. The term "similar" when applied to two
non-numerical characteristics means that the two characteristics
resemble each other, or share common properties.
[0090] As used herein, the term "substantial" means more than a
minimal or insignificant amount; and the term "substantially" means
more than minimally or insignificantly.
[0091] As used herein, the terms "substantially equal" and
"substantially the same" when applied to numerical values means
that two or more numerical values are very close in value to each
other, e.g., a first value differs from a second value by no more
than about 10% of the first value, or by no more than about 5%, or
by no more than about 3%, or by no more than about 2%, or by no
more than about 1%. The terms "substantially equal" and
"substantially the same" when applied to two non-numerical
characteristics means that the two characteristics differ from one
another only in insignificant ways, or are the nearly the same as
each other, or are identical.
[0092] The terms "blood" and "whole blood" refer to blood as it
exists within an animal and as directly obtained from a subject in
a blood sample. Blood contains red blood cells, white blood cells,
proteins such as albumin, globulins, and clotting factors, salts,
water, and other constituents.
[0093] The terms "plasma" and "blood plasma" refer to the liquid
portion of blood (e.g., a blood sample) that remains after the
removal of blood cells. Red blood cells and white blood cells may
be removed by centrifugation of a blood sample, leaving plasma
above the pelleted cells in the bottom of the centrifuge tube.
Plasma retains blood clotting factors, and is obtained from
anti-coagulated blood samples.
[0094] The terms "serum" and "blood serum" refer to the liquid
portion of blood that remains after blood is allowed to clot, and
the clot is removed. Serum differs from plasma in that serum lacks
clotting factors: since clotting requires fibrin, thrombin, and
other proteins, which form and remain part of a blood clot, serum
lacks these proteins while plasma contains them.
[0095] As used herein, "vitamin D" refers to vitamin D in all its
forms, e.g., vitamin D1 through vitamin D5, including without
limitation vitamin D2 (ergocalciferol), vitamin D3
(cholecalciferol), and hydroxy forms of vitamin D. The major
circulating forms of vitamin D are 25-hydroxy vitamin D3 and
25-hydroxy vitamin D2.
[0096] As used herein, the term "Relative Light Units" and its
abbreviation "RLU" refer to the units used to scale the output of
instruments which measure light intensity (e.g., a photomultiplier
tube, luminometer, or other such instrument or device). RLU values
are typically proportional to photon number; reporting light
intensity in RLU allows comparison between experiments and between
instruments.
[0097] As used herein, the term "coefficient of variance" and its
abbreviations "COV" and "CV" are used as commonly understood in the
art; COV values are typically reported as a percent. The COV is
calculated by dividing the standard deviation (SD) of a set of
experimental values by the mean value (M) of that set of
experimental values to obtain the ratio of the standard deviation
to the mean, and then multiplying that ratio by 100:
COV=SD/M.times.100
The COV provides a measure of the differences between observed
measurement values where multiple experiments and measurements are
made; where multiple experiments give widely varying results, the
COV is large; where multiple experiments give results that are all
closely matched, the COV is small. A small COV for the results of
an experimental procedure indicates that the experimental procedure
provides consistent results; this is typically interpreted to mean
that a small COV indicates better results than a larger COV.
[0098] As used herein, a "binding compound", a "binding molecule"
and a "binding protein" all refer to molecules that bind to a
target molecule. For example, an antibody or antibody fragment
which specifically binds vitamin D2 may be termed a vitamin D2
binding molecule, or a vitamin D2 binder; and an antibody or
antibody fragment which specifically binds vitamin D3 may be termed
a vitamin D3 binding molecule, or a vitamin D3 binder. In some
instances, the same molecule may bind more than one target
molecule; this may be true for antibodies and antibody fragments as
well as for other binders. Thus, for example, an antibody or
antibody fragment which specifically binds vitamin D2 and vitamin
D3 (e.g., binds D2 and D3 forms of vitamin D, but does not
significantly bind other target molecules that are not forms of
vitamin D) may be termed a vitamin D binding molecule; a vitamin D
binder; a vitamin D2/D3 binding molecule; a vitamin D2/D3 binder; a
vitamin D2 and D3 binding molecule; a vitamin D2 and D3 binder; or
other similar term.
[0099] As used herein, the term "binder" refers to a binding
molecule as discussed above. In particular, a "vitamin D binder" is
a compound that specifically binds to vitamin D in any of its
forms. A vitamin D binder may be an antibody, an antibody fragment,
an aptamer, a binding protein, a receptor, an immunoadhesin, a
small molecule having affinity for vitamin D, or any compound that
binds to one or more forms of vitamin D.
[0100] As used herein, the term "bound" means that two compounds
are in a tight, non-covalent, interaction with one another. Each
compound is bound to the other. Binding may be specific: for
example, when a ligand is in contact with its receptor, the ligand
is said to be bound to the receptor, and when an antigen is in
contact with an antibody specific for that antigen, the antigen is
said to be bound to the antibody. The term "bound" is typically
used to refer to specific binding between binding partners.
Compounds which bind are said to "associate" with each other, and
the rate of binding is the association rate.
[0101] As used herein, the term "unbound" refers to a compound that
is not in a tight, non-covalent, interaction with another compound.
Typically, the term "unbound" refers to the lack of specific
binding between potential binding partners. For example, where
antigens and antibodies are present in a solution, an antigen
molecule that is free in the solution is said to be an unbound
antigen. The rate of unbinding is the dissociation rate.
[0102] It will be understood that binding between partners may be
transient, so that there may be continuous binding and unbinding
between compounds that bind each other; however, an equilibrium may
be reached where the numbers of bound and unbound compounds remain
substantially constant for a period of time.
[0103] As used herein, the term "competitive binding" refers to
situations where multiple potential binding partners are present in
the same solution, where some potential binding partners bind
together and others remain free in solution. The multiple binding
partners may include a single population of substantially identical
compounds (e.g., ligands or antigens) which may bind with a target
binding partner (e.g., a receptor or antibody), or may include two
or more populations of potential binding partners (e.g., ligands or
antigens), each of which population may bind with a target binding
partner (e.g., a receptor or antibody). For example, where multiple
antigens bind a single antibody, the antigens are said to
"cross-react" with the antibody, and may be termed
"cross-reactants" with respect to that antibody. In some cases, one
population which binds may be labeled, and another population may
be unlabeled.
[0104] As used herein, the terms "competition assay", "competition
methods", "competitive binding assays" and the like refer to assays
and methods in which an analyte and a labeled molecule that is
believed to be nearly identical, or at least similar, to the
analyte (with respect to its binding characteristics) are present
in a solution with a molecule which binds both the analyte and the
labeled molecule (e.g., an antibody). Competitive binding assays
rely on the ability of a labeled molecule (such as, e.g., labeled
vitamin D) to compete with the analyte (such as, e.g., vitamin D)
for binding with a limited amount of binder (e.g., an antibody,
such as an anti-vitamin D antibody). The amount of analyte in the
test sample is inversely proportional to the amount of labeled
molecule that becomes bound to the molecule which binds both the
analyte and the labeled molecule.
[0105] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the analyte to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three part complex (see, e.g.,
U.S. Pat. No. 4,376,110). The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays), or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay (enzyme linked
immunosorbent assay), in which case the detectable moiety is an
enzyme. Useful for detecting proteins and other large target
compounds, sandwich ELISA assays are typically not used for the
detection of small molecules.
[0106] As used herein, the term "antibody" is used in the broadest
sense and specifically covers single monoclonal antibodies
(including agonist and antagonist antibodies) and antibody
compositions with polyepitopic specificity. Thus, antibodies may be
polyclonal antibodies, e.g., may be antibodies purified from the
blood of an animal such as a sheep or goat which has been
challenged by a target antigen, and may be monoclonal antibodies.
For example, monoclonal antibodies may be made by the hybridoma
method first described by Kohler et al., Nature, 256:495 (1975), or
may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567 to Cabilly et al.). The "monoclonal antibodies" also
include clones of antigen-recognition and binding-site containing
antibody fragments (Fv clones) isolated from phage antibody
libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example. Monoclonal antibodies are obtained
from a population of substantially homogeneous antibodies, i.e.,
the individual antibodies comprising the population are identical
except for possible naturally-occurring mutations that may be
present in minor amounts.
[0107] Antibodies (e.g., IgG antibodies) are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light chains (LCs) and two identical heavy chains
(HCs). Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges between cysteines. Each heavy chain has at a
variable domain, followed by a number of constant domains. The
variable domains are disposed closer to the amino-terminal
(N-terminal) portion of the HC than are the constant domains;
conversely, the constant domains are disposed closer to the
carboxy-terminal (C-terminal) portion of the HC than are the
variable domains. Similarly, each light chain has a variable domain
at one end (towards the N-terminal) and a constant domain at its
other end (towards the C-terminal); the constant domain of the
light chain is aligned with the first constant domain of the heavy
chain, and the light chain variable domain is aligned with the
variable domain of the heavy chain. Particular amino acid residues
are believed to form an interface between the light- and
heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651
(1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592
(1985)). The variable domains form the antigen-binding sites; thus
an intact antibody has two antigen binding sites composed of
variable domains of the LC and HC pairs.
[0108] "Antibody fragment", and all grammatical variants thereof,
as used herein are defined as a (1) portion of an intact antibody
comprising the antigen binding site or variable region of the
intact antibody, wherein the portion is free of the constant heavy
chain domains (i.e. CH2, CH3, and CH4, depending on antibody
isotype) of the Fc region of the intact antibody, and (2)
constructs comprising a portion of an intact antibody (as defined
by the amino acid sequence of the intact antibody) comprising the
antigen binding site or variable region of the intact antibody.
Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2,
Fd, Fc, Fv, diabodies, and any other "Non-single-chain
antigen-binding unit" as described, e.g., in U.S. Pat. No.
7,429,652. The term "intact antibody" refers to the complete
antibody, or the amino acid sequence of the complete antibody, of
which an antibody fragment is a part. It will be understood that an
antibody fragment may be produced by partial digestion (e.g., by
papain or pepsin) of an intact antibody, or may be produced by
recombinant or other means.
[0109] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain connected to a light chain variable domain in the
same polypeptide chain. By using a linker that is too short to
allow pairing between the two domains on the same chain, the
domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, for example, EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448
(1993).
[0110] As used herein, an "antigen-binding antibody fragment" is
any antibody fragment that retains the ability to bind to the
specific target to which the intact antibody specifically binds. An
antigen-binding antibody fragment may have different (e.g., lesser)
binding affinity for the target antigen than the intact
antibody.
[0111] The term "monoclonal antibody" (mAb) as used herein refers
to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally
occurring mutations that may be present in minor amounts. For
example, monoclonal antibodies may be made by the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567
to Cabilly et al.). The "monoclonal antibodies" also include clones
of antigen-recognition and binding-site containing antibody
fragments (Fv clones) isolated from phage antibody libraries using
the techniques described in Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0112] Nom A "secondary antibody" is an antibody used to bind other
antibodies; these other antibodies are termed primary antibodies.
Secondary antibodies may be used, for example, to bind an
antigen-binding antibody to a capture surface. For example, where a
primary antibody is used to recognize a target antigen, a secondary
antibody may be used to bind the primary antibody to a capture
surface. Any antibody, from any source, including a polyclonal
antibody, a monoclonal antibody, an antibody fragment, an antibody
derivative, and combinations of these, may be used as a secondary
antibody. In some instances, a primary antibody may be, e.g., a
sheep antibody (an antibody raised in sheep), and a secondary
antibody may be, e.g., a rabbit antibody (an antibody raised in
rabbits).
[0113] An "anti-sheep antibody" is an antibody that binds sheep
antibodies, typically by recognizing an epitope on the Fc portion
of the sheep antibody. For example, in some examples disclosed
herein, vitamin D (both labeled and unlabeled) is bound by a sheep
antibody; that sheep antibody is itself bound by an "anti-sheep
antibody" which recognizes an epitope on the vitamin-D-binding
sheep antibody, and (as disclosed herein) such binding may be used
to attach the bound vitamin D to a capture surface. The anti-sheep
antibodies used in the examples disclosed herein are rabbit
polyclonal antibodies raised against the Fc portion of sheep IgG
antibodies; however, any antibody, whether polyclonal or
monoclonal, rabbit, or other animal, that binds sheep antibodies
may be used to bind sheep antibodies to a capture surface.
[0114] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or, an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope.
[0115] As used herein, the term "aptamer" refers to a nucleic acid
molecule capable of binding to a target molecule. The nucleic acid
may be a deoxyribonucleic acid, a ribonucleic acid, a linked
peptide nucleic acid, or other nucleic acid, analog, or derivative
thereof. The generation and use of aptamers is known in the art;
see, e.g., U.S. Pat. No. 5,475,096.
[0116] An immunoadhesin is an antibody-like chimeric molecule
comprising a portion of an antibody (e.g., some or all of the Fc
region of an IgG antibody) linked to a receptor or ligand having
affinity for a target molecule. Immunoadhesins combine the binding
specificity of a heterologous protein (an "adhesin") with the
effector functions of immunoglobulin constant domains.
Structurally, the immunoadhesins comprise a fusion of an amino acid
sequence with the desired binding specificity which is other than
the antigen recognition and binding site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence.
The adhesin part of an immunoadhesin molecule typically is a
contiguous amino acid sequence comprising at least the binding site
of a receptor or a ligand. The immunoglobulin constant domain
sequence in the immunoadhesin may be obtained from any
immunoglobulin. Immunoglobulin fusions are discussed, for example,
in U.S. Pat. No. 5,428,130.
[0117] As used herein, the term "small molecule" refers to a
compound, typically a non-polymeric organic compound, that is
smaller than a typical protein. Examples of small molecules include
acetylsalicylic acid (aspirin), caffeine, cholesterol, vitamin D,
and other molecules.
[0118] The terms "antigen", "target molecule", "target
polypeptide", "target epitope", and the like are used herein to
denote the molecule (or, regarding an epitope, portion of molecule)
specifically bound by a binder such as an antibody, antibody
fragment, or other binder. For example, vitamin D is the target
molecule for an anti-vitamin D antibody or antigen-binding fragment
thereof.
[0119] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (K.sub.d).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention.
[0120] The word "label" or "detectable label" when used herein
refer to a detectable compound or composition which is linked
(e.g., conjugated) directly or indirectly to a compound so as to
generate a "labeled" compound. The label may be detectable by
itself (e.g. radioisotope labels or fluorescent labels) or, in the
case of an enzymatic label, may catalyze chemical alteration of a
substrate compound or composition which is detectable. A label may
serve as a substrate for an enzyme, or may otherwise participate in
a reaction. A label may aid in binding a compound to another
compound or to a substrate. A label may be, for example, selected
from the group consisting of a dye, an epitope tag, a fluorescent
moiety, a luminescent moiety, a chemiluminescent moiety, an
enzymatic label, a magnetic label, a paramagnetic label, a contrast
agent, a nanoparticle, a radioisotope, biotin, streptavidin, and a
quencher.
[0121] For example, a label may be an alkaline phosphatase label,
in which the results of a reaction catalyzed by alkaline
phosphatase is observed, and may be used to identify an analyte or
verify its presence in a sample, and may be used to quantify an
analyte in a sample. Alkaline phosphatase reagents are commercially
available; for example, Nitroblue Tetrazolium (NBT) is used with
the alkaline phosphatase substrate 5-Bromo-4-Chloro-3-Indolyl
Phosphate (BCIP) to provide a colored product which may be observed
and quantitated. Other reagents include Fast Red TR/Naphthol AS-MX
and TR phosphate
(4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid
2,4-dimethylanilide phosphate, reagents for the production of
p-nitrophenol, and others.
[0122] For example, a label may be a peroxidase label (such as
horseradish peroxidase, myeloperoxidase, or other peroxidase) in
which the results of a reaction catalyzed by the peroxidase is
observed, and may be used to identify an analyte or verify its
presence in a sample, and may be used to quantify an analyte in a
sample. Benzidine-containing compounds (e.g., diaminobenzidine,
tetramethyl benzidine), aniline-containing compounds,
aminoantipyrene compounds, Trinder reagents, and other reagents
known in the art may be used to provide a detectable product in the
presence of a peroxidase.
[0123] A label may be a dye, such as rhodamine and related
rhodamine dyes (e.g., tetramethylrhodamine carboxytatramethyl
rhodamine (TAMRA), and others), fluorescein and fluorescein
derivatives (e.g., 5-carboxyfluorescein, 6-carboxy fluorescein and
others), phycoerythrin, umbelliferone, Texas Red, rare earth
chelates (europium chelates), dansyl dyes (including, e.g.,
dansylamide dyes, dansyl cadaverine, dansyl chloride, and others);
cyanine dyes (e.g., Cy3, Cy5, SYBR green, and others); Lissamine;
phycoerythrins; Texas Red; and analogs thereof.
[0124] A label may be a fluorescent material, including fluorescent
dyes, and including green fluorescent protein and other fluorescent
proteins known in the art. A label may be a luminescent moiety,
such as luminol, or other luminescent material, including
bioluminescent materials such as luciferase, luciferin, and
aequorin.
[0125] A label may be a nanoparticle, such as a gold nanoparticle
(e.g., a colloidal gold particle), or a quantum dot (e.g., a small
particle, typically a semiconductor, which may be detectable upon
application of an appropriate amount and wavelength of
electromagnetic radiation, e.g., by illumination). A label may be a
magnetic label, or a paramagnetic label, which may be a
nanoparticle or bead. A label may be a radioisotope or other
radioactive material, including, e.g., .sup.131I, .sup.125I,
.sup.111In, .sup.99TC, .sup.35S, .sup.14C, and .sup.3H.
[0126] As used herein, a "capture element" is used to link a
molecule to another molecule or to a support, such as a solid
support. A capture element may be, for example, a polynucleotide, a
polypeptide, a peptide nucleic acid, a locked nucleic acid, an
oligosaccharide, a polysaccharide, an antibody, an antibody
fragment, an antibody mimic, an immunoadhesin, a receptor, a
ligand, a lipid, biotin, avidin, streptavidin, Extravidin,
neutravidin, or other avidin derivative or avidin analog, a metal,
an epitope tag, and any portion of any of these. A capture element
binds to a binding partner, such as a tag; e.g., where the capture
element is an antibody or antibody fragment, the tag is the
corresponding antigen to which the antibody or antibody fragment
specifically binds. For example, a capture element may include a
biotin group or a biotin binding protein such as, e.g., avidin,
streptavidin, neutravidin, or Extravidin. In further examples, a
capture element may be either polyhistidine or nitrilotriacetic
acid (NTA) chelated with a metal ion such as nickel, cobalt, iron,
or any other metal ion able to form a coordination compound with
poly-histidine when chelated with NTA. A capture element may be or
include a polynucleotide that is designed to hybridize directly
with a complementary nucleic acid sequence.
[0127] As used herein, a "capture surface" is a solid surface,
which may be free floating (e.g., a bead) or may be attached to, or
part of, a structure (e.g., a wall of a tube, or a side of a dish
or flask). A capture surface may include capture elements effective
to link a labeled compound to the capture surface. For example, a
capture surface may comprise avidin where a labeled compound
comprises biotin; may comprise an antibody (e.g., an anti-sheep
antibody, where the antibody that binds the target analyte is a
sheep antibody); a combination of these; and may comprise any other
agent, or any combination of these or other agents, effective to
link a compound to a capture surface.
[0128] As used herein, a "low pH" refers to a pH of between about
pH 2 to about pH 6, or, in some instances, a pH range of between
about pH 2.5 to about pH 5.5, or more particularly, a pH range of
between about pH 3 to about pH 5, or between about pH 2.5 to about
pH 4.
[0129] As used herein, a "neutral pH" refers to a pH of between
about pH 6 to about pH 9, or, in some instances, a pH range of
between about pH 6.5 to about pH 8.5, or more particularly, to a pH
range of between about pH 7 to about pH 8.
[0130] As used herein, the term "aspartyl peptidase" refers to a
protelytic enzyme with an active site containing an aspartate
residue. Aspartyl pepidases are classified within Enzyme Commission
(EC) number EC 3.4.23 (e.g., pepsin A (EC 3.4.23.1), pepsin B (EX
3.4.23.2), pepsin C or gastricsin (EC 3.4.23.3) and chymosin (EC
3.4.23.4)). Aspartyl peptidases include digestive, lysozomal,
bacterial, fungal, viral and other proteases. Aspartyl peptidases
typically are most active under acidic conditions, such as, for
example, a pH range of between about pH 2 to about pH 6, or, in
some instances, a pH range of between about pH 3 to about pH 5, or
other acidic pH range. Aspartyl peptidases include, without
limitation, for example, pepsin (including pepsin A, B, and C),
gastricsin, chymosin (rennin), cathepsin, renin, HIV protease,
plasmepsin, retropepsin, and nepenthesin. A more particular, but
not exhaustive, list of aspartyl peptidases includes pepsin A (Homo
sapiens), cathepsin D (Homo sapiens), nepenthesin (Nepenthes
gracilis), walleye dermal sarcoma virus retropepsin (walleye dermal
sarcoma virus), Ty3 transposon peptidase (Saccharomyces
cerevisiae), Gypsy transposon peptidase (Drosophila melanogaster),
Osvaldo retrotransposon peptidase (Drosophila buzzatii),
retrotransposon peptidase (Schizosaccharomyces pombe),
retrotransposon 17.6 peptidase (Drosophila melanogaster),
cauliflower mosaic virus-type peptidase (cauliflower mosaic virus),
bacilliform virus peptidase (rice tungro bacilliform virus),
thermopsin (Sulfolobus acidocaldarius), signal peptidase II
(Escherichia coli), spumapepsin (human spumaretrovirus), Copia
transposon peptidase (Drosophila melanogaster), Ty1 transposon
peptidase (Saccharomyces cerevisiae), presenilin 1 (Homo sapiens),
impas 1 peptidase (Homo sapiens), and yeast proteinase A.
[0131] As used herein, the term "aspartyl peptidase active at low
pH" refers to an aspartyl peptidase that is active within a pH
range of between about pH 2 to about pH 6, or, in some instances, a
pH range of between about pH 3 to about pH 5, or between about pH
2.5 to about pH 4. An aspartyl peptidase active at low pH is
inactivated (i.e., its activity is reduced significantly reduced)
at neutral and basic pH. For example, the peptidase activity of an
aspartyl peptidase active at low pH is reduced by greater than
about 50%, or by greater than about 75%, at pH 8.
[0132] As used herein, an enzyme (e.g., such as a peptidase) is
"active" or "activated" when, in the presence of sufficient
substrate, the rate or extent of a reaction catalyzed by the enzyme
is at or near normal levels, and an enzyme (e.g., such as a
peptidase) is "inactive" or "inactivated" when, in the presence of
sufficient substrate, the rate or extent of a reaction catalyzed by
the enzyme is reduced significantly below normal levels, or is
negligible or absent.
[0133] As used herein, a "finger-stick" refers to: i) the act of
making a small puncture in the skin of a subject, allowing a small
amount (e.g., a droplet, or one, two, or a few drops) of blood to
flow and become available for collection; ii) the puncture itself;
and iii) the blood collected thereby. Blood may be liberated in a
finger-stick, for example, by use of a lancet or other sharp
implement effective to pierce the skin of a subject. Typically,
only a small amount of blood is collected in this way (e.g., the
amount of blood may be about 250 .mu.L or less, or about 200 .mu.L
or less, or about 150 .mu.L or less, or about 100 .mu.L or less, or
about 50 .mu.L or less, or about 25 .mu.L or less, or about 15
.mu.L or less, or about 10 .mu.L or less, or about 10 .mu.L or
less, or about 5 .mu.L or less, or about 3 .mu.L or less, or about
1 .mu.L or less). Blood from a finger-stick may be collected, e.g.,
by needle, syringe, capillary tube, or other method. Blood from a
finger-stick may be collected for transport to another location;
for storage prior to use or analysis; for immediate use; or for a
combination of the same.
[0134] Assay methods disclosed herein provide fast assays, in part
because denaturation in acid is fast and allows the aspartyl
peptidase (e.g., pepsin) to digest vitamin D binding protein
rapidly. In addition, although vitamin D binding protein has a high
affinity for vitamin D and so could compete with an antibody for
vitamin D, degradation of the binding protein by the aspartyl
peptidase rapidly destroys the ability of the binding protein to
bind vitamin D, and so soon reduces and eliminates such
competition, providing a rapid and accurate assay of vitamin D
levels.
[0135] Measurement of free vitamin D may be made using molecules
which specifically bind to vitamin D or to a particular species of
vitamin D (for example, anti-vitamin D antibodies such as
anti-25-hydroxy vitamin D antibodies). As noted previously, the
major forms of vitamin D circulating in blood are 25-hydroxy
vitamin D3 and 25-hydroxy vitamin D2. Since levels of different
forms of vitamin D are clinically relevant (e.g., D2 and D3 forms
of vitamin D), different binders may be used in detecting vitamin D
and in measuring vitamin D levels. Thus, for example, in
embodiments, a binding molecule for 25-hydroxy vitamin D3 and a
binding molecule for 25-hydroxy vitamin D2 may be used, alone or
together, in assays and methods as disclosed herein. In
embodiments, molecules which bind more than one form of vitamin D
may be used. For example, a binding molecule for 25-hydroxy vitamin
D3 and a binding molecule for 25-hydroxy vitamin D2 may be used,
alone or together, in assays and methods as disclosed herein. A
binding molecule that binds both 25-hydroxy vitamin D2 and
25-hydroxy vitamin D3 may be used. Mixtures of these, and other
such binders, may be used in assays as disclosed herein. In
embodiments, other binders, including other antibodies, e.g.,
antibodies to non-hydroxylated vitamin D, to di-hydroxylated
vitamin D, etc., may be used.
[0136] In embodiments, assays are provided in which binders (e.g.,
antibodies) specific for vitamin D are used to determine the
amounts of vitamin D in a sample. For example, in embodiments,
assays are provided in which binders (e.g., antibodies) specific
for a 25-hydroxy form or forms of vitamin D are used to determine
the amounts of vitamin D in a sample. In embodiments where a binder
specific for one particular form of vitamin D is used, assays
specific for that form of vitamin D are provided. For example,
where a binder specific for 25-hydroxy vitamin D3 is used, the
methods disclosed herein may be used to quantify vitamin D3 in a
sample. Multiple binders may be used together; e.g., a binder
specific for 25-hydroxy vitamin D2 and a binder specific for
25-hydroxy vitamin D3 may be used together to quantify 25-hydroxy
vitamin D2 and 25-hydroxy vitamin D3 forms of vitamin D. For
example, assays disclosed herein may use antibodies which bind both
25-hydroxy vitamin D2 and 25-hydroxy vitamin D3, albeit with
unequal affinities, in amounts so as to provide substantially the
same detection for both 25-hydroxy vitamin D2 and 25-hydroxy
vitamin D3.
[0137] In embodiments, assays may use VDBP as the binder to measure
levels of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3, or other
forms of vitamin D. In addition, embodiments of the methods and
assays disclosed herein include any other assay where an analyte is
bound to an endogenous binding protein or set of binding proteins
or other "binder" where the analyte is released and may be
quantified following denaturation and enzymatic degradation of the
binding protein by use of an aspartyl peptidase under acidic
conditions.
[0138] Embodiments of the methods and assays disclosed herein
include embodiments utilizing any proteolytic enzyme that is active
under conditions likely to denature the vitamin D binding protein,
where the proteolytic enzyme can be inactivated (e.g., becomes
inactive at neutral pH, upon dilution, or heating, or other
condition).
[0139] In embodiments of the methods and assays disclosed herein,
reagents may be provided in liquid form. In embodiments, liquid
reagents may be provided in pre-filled, sealed containers ready for
use in an assay when needed. In embodiments, liquid reagents
provided in pre-filled, sealed containers may be sterile reagents,
and the pre-filled, sealed containers may be sterile
containers.
[0140] In embodiments of the methods and assays disclosed herein,
reagents may be provided in dry form (e.g., may be lyophilized, or
provided as a powder, or as a cake, or other solid form). In
embodiments, dry reagents may be provided in pre-filled, sealed
containers ready for use in an assay when needed. In embodiments,
dry reagents provided in pre-filled, sealed containers may be
sterile reagents, and the pre-filled, sealed containers may be
sterile containers. In embodiments, containers with dry reagents
may be configured for introduction of liquid effective to dissolve
a dry (e.g., a lyophilate, a powder, a cake, or other solid)
reagent.
[0141] In embodiments of the methods and assays disclosed herein, a
capture surface may be preformed and dried. Such a capture surface
may be prepared, for example, using methods disclosed in U.S. Pat.
No. 8,088,593. In addition, pepsin may be dried (lyophilized) into
a container, such as a tube, caplet, vial, cuvette, or other
container; the container may subsequently be sealed. Dried pepsin
may be dissolved in an appropriate solution or solvent, such as,
e.g., 100 mM citrate buffer (about pH 2 to 3, e.g., pH 2.6) prior
to use in an assay. Other reagents used in assays as disclosed
herein may also be sealed into containers (such as tubes) and
packed together into a kit for use in an assay as disclosed herein.
Such a kit may be sealed, labeled and bar-coded prior to shipping
to customers and other users.
[0142] In embodiments, a blood sample may be obtained from a
subject by performing a finger-stick, and collecting blood
liberated thereby from the subject. Typically, only a drop or only
a small amount of blood is collected in this way. Blood from a
finger-stick may be collected, e.g., by needle, syringe, capillary
tube, or other method. For example, when collected by capillary
tube, an outer end of the glass capillary may be touched to the
blood droplet so that blood is drawn by capillary action into the
capillary tube for use in analysis by flow to a collection or
analysis device, or by transfer of the tube itself, or for analysis
within the tube.
EXAMPLES
[0143] The following examples provide descriptions of embodiments
of the methods disclosed herein. These methods of measuring vitamin
D were applied to clinical blood samples, including plasma, serum,
and whole blood, as discussed below. The following list and
description of materials describes some of the materials used in
performing the assays and measurements described in the following
exemplary methods.
[0144] "TRIS.RTM.-buffered saline" (TBS) was
(Tris(hydroxymethyl)aminomethane in saline, supplied as 10.times.
solution; when diluted, TBS contained about 20 mM TRIS.RTM. and
0.9% NaCl, at about pH 7.4. TBS was obtained from Sigma.
[0145] "Low BSA 0.03% in TB S" was 0.03% bovine serum albumin (BSA)
and "3% BSA in TBS" was 3% BSA (Fraction V, 99% Pure) in 50 mM TBS,
with 0.05% Sodium Azide, pH 8.0 (Sigma Catalog #3059).
[0146] "Wash buffer" was 50 mM TBS, 0.05% Tween 20, 0.05% Sodium
azide, pH 8.
[0147] Other buffers (e.g., carbonate-bicarbonate buffer) and
reagents (e.g., citric acid and sodium citrate) were also obtained
from Sigma. For example, citric acid (anhydrous) is Sigma catalog
#251275, and sodium citrate (dihydrate) is Sigma catalog
#S1804.
[0148] Citrate buffer was made as follows: 900 mL of HPLC grade
water was added to 17.77 g Citric Acid (anhydrous) and 4.27 g
Sodium Citrate (dihydrate), and the solution was mixed until the
solids were dissolved; the pH was checked with a calibrated pH
meter. If the measured pH was not within the range of pH 2.4 to pH
3.0, the solution was discarded. The pH was adjusted (when
necessary) to 2.6 using 1N NaOH or HCl, while on a mixing stand. If
pH adjustment led to overshooting of the pH, the buffer was
discarded. Upon adjustment to pH 2.6, water was added to provide a
final volume of 1 L buffer.
[0149] The following buffers were obtained from Pierce: "Super
Block", a TRIS.RTM.-buffered saline, pH 7.4, with Kathon
preservative and containing a proprietary protein formulation
(Pierce Catalog #37535); "Starting Block", a TBS (Pierce Catalog
#37542); "Sea Block", a PBS (Pierce Catalog #37527); and "Protein
Free", a TBS lacking protein (Pierce Catalog #37570).
[0150] 25-hydroxy vitamin D3 and 25-hydroxy vitamin D2 were
obtained from Sigma (Sigma-Aldrich Corporation, Saint Louis, Mo.).
Vitamin D conjugates were obtained from Dojindo (Dojindo Molecular
Technologies, Rockville, Md.).
[0151] Anti-vitamin D antibodies were obtained from commercial
suppliers. A monoclonal antibody directed to 25-hydroxy vitamin D3
(catalog no. ABIN108769) was obtained from Antibodies-Online
(Atlanta, Ga., USA). A monoclonal antibody directed to 25-hydroxy
vitamin D3 (catalog no. MAB6566) was obtained from R&D Systems
(Techne Corp., Minneapolis, Minn., USA). Sheep anti-vitamin D
antibodies were obtained from Bioventix (Sheep Monoclonal Anti
25-Hydroxyvitamin D Antibodies, Bioventix catalog no.
BVX.vitD3.5H10 ("Ab5H10"); catalog no. BVX.vitD2.2D10 ("Ab2D10");
and catalog no. BVX.vitD3.2F4 ("Ab2F4"); Bioventix Inc., Farnham,
Surrey, U.K.). Other antibodies were obtained from Fitzgerald
(biotin-conjugated rabbit anti-Sheep IgG (Fc), catalog no.
43C-CB1326, a rabbit polyclonal antibody that binds to epitopes on
the Fc portion of sheep IgG antibodies); Fitzgerald Industries
International, Inc., Acton, Mass.).
[0152] Pepsin was porcine gastric mucosa pepsin (Sigma catalog
#P7000) unless otherwise stated.
Example 1: Total 25-Hydroxy Vitamin D Assay
[0153] A biotin-labeled anti-sheep antibody coated on
UltraAvidin.TM. served as the capture surface. Tests were performed
with a final antibody concentration of 50 ng/mL and biotin
conjugate of 1:10,000 from stock. Super Block (Pierce Catalog
#37535) produced the best modulation.
[0154] Alkaline Phosphatase Conjugate
[0155] A custom 25-hydroxy vitamin D3 alkaline phosphatase (AP)
conjugate was tested in the assay system with the following format:
biotin-labeled anti-sheep antibody surface, with the unlabeled
analyte competing with the AP labeled analyte unlabeled sheep
anti-25-hydroxy vitamin D antibody in solution. This format and
conjugate was used for initial assay screening and a final sample
dilution of 1:5 was used to allow comparison between methods as
disclosed herein using the custom conjugate and the commercial
25-hydroxy vitamin D biotin conjugate. The custom 25-hydroxy
vitamin D-AP conjugate with the anti-sheep surface was validated
for use in the present assay methods. StabilZyme.RTM. AP Conjugate
Stabilizer (SurModics, Eden Prairie, Minn.55344, USA, Stock Code
SA01-0050) was chosen as a suitable alkaline phosphatase
stabilizing diluent.
Pretreatment of Serum or Plasma Samples
[0156] Pepsin was lyophilized in a reagent tube and reconstituted
(dissolved) with citrate buffer during the assay procedure. (Pepsin
from porcine gastric mucosa and from other sources yielded similar
results.) The pepsin was lyophilized at 480 .mu.g per reaction
tube, the automated protocol moved 48 .mu.L of citrate buffer into
the tube, which was then dissolved and mixed prior to use. The
results obtained using reconstituted, previously lyophilized pepsin
and liquid pepsin were substantially equivalent.
[0157] Serum and plasma samples were diluted 1:5 into a solution of
10 mg/mL pepsin (in 100 mM citrate buffer at pH 2.6) and incubated
for 5 minutes at 34.degree. C. To stop the pepsin digestion and
inactivate the pepsin, the reaction mixture was further diluted
1:10 into 50 mM TRIS pH 8.0 (final sample dilution 1:50). The
resulting pretreated diluted sample solution can be used
immediately in an immunoassay for vitamin D.
Pepsin Sample Treatments and Assay of Vitamin-D
[0158] In this example, anti-25-hydroxy vitamin D antibodies were
used to detect vitamin D. Antibodies to other forms, or to multiple
forms, of vitamin D may also be used. Binders other than antibodies
may be used.
[0159] Pepsin was used to free vitamin D from VDBP for quantitative
measurements of vitamin D in blood samples. Use of pepsin has not
been reported for vitamin D (e.g., 25-hydroxy vitamin D) assays.
Binding of 25-hydroxy vitamin D to VDBP would be expected to
decrease at acidic pH, a pH range in which pepsin is active and
will digest VDBP. Thus, the combination of very low pH and the
digestion by pepsin work synergistically to enhance the release of
25-hydroxy vitamin D and thus to improve the speed and accuracy of
vitamin D measurements.
[0160] However, low pH alone did not provide optimal conditions for
the assays and methods disclosed herein. Use of strong acid alone,
such as HCl, was found to cause precipitation and clumping of the
sample plus reagent solutions, making vitamin D measurements
difficult. In addition, use of strong acid alone, such as HCl, did
not provide stable or repeatable final pH values following sample
addition. It was surprisingly found that low pH buffered solutions,
such as citrate buffered solutions (e.g., citrate buffers of about
pH 2.4 to about pH 3.0, preferably pH 2.6) provided favorable
conditions for aspartyl peptidase denaturation and degradation of
VDBP for the release of vitamin D, while providing stable and
repeatable pH values following sample addition. Accordingly,
Applicants disclose herein aspartyl peptidase-based methods for
assaying vitamin D levels in blood samples that utilize low pH
buffered solutions, including low pH citrate buffers.
[0161] Samples were pre-treated as described above. The assay
system and general method used are as described above, and may use
devices, systems, and methods as described in U.S. Pat. No.
8,088,593; in U.S. Pat. No. 8,380,541; in PCT/US012/57155, filed
Sep. 24, 2012; and in U.S. patent application Ser. No. 13/244,947,
filed Sep. 26, 2011. Reagents were: Pepsin: the pepsin solution was
10 mg/mL porcine pepsin in 100 mM citrate buffer pH 2.6 (final pH
3.9 following addition of pepsin). (The pepsin used was from
porcine gastric mucosa; other experiments confirmed that similar
results were obtained with pepsin from other sources.) Unless
otherwise stated, the pepsin was lyophilized at 480 .mu.g per
reaction tube, the automated protocol moved 48 .mu.L of citrate
buffer into the tube, which was then dissolved and mixed prior to
use. Labeled Conjugate: Alkaline phosphatase (calf
intestine)-labeled 25-hydroxy-vitamin-D3 (concentration estimated
at about 10 ng/mL with a hapten/enzyme ratio of 4:1). The
chemiluminogenic alkaline phosphatase substrate was Phospho-Glo
Substrate (KPL). Wash: The Wash buffer was 50 mM TBS, 0.05% Tween
20, 0.05% Sodium azide, pH 8. Substrate: Polystyrene reaction
vessels, coated with UltraAvidin.TM. as described in U.S. Pat. No.
8,088,593, were used for these assays. (During manufacturing:
UltraAvidin.TM.-coated tips were exposed first to biotinylated
rabbit antibody then washed and "blocked" with a commercially
available blocking buffer.)
[0162] The liquid phase of the assay reaction included the sample
(mixed with the sample diluent) and included the anti-vitamin-D
antibodies and the alkaline phosphatase (AP) conjugate. As
discussed below, in many cases mixtures of two types of
anti-vitamin D antibodies were used. For example, in many cases,
the anti-vitamin-D antibodies Ab5H10 and Ab2F4 were mixed into the
diluted sample (final concentrations 10 ng/mL Ab5H10 and 5 ng/mL
Ab2F4), and then the AP conjugate was mixed into the solution that
included the sample, the sample diluent, and the anti-vitamin-D
antibodies. This liquid mixture was incubated with the capture
surface (anti-sheep antibody bound to UltraAvidin.TM.-coated
polystyrene).
[0163] After extensive optimization of buffer, pH, concentration
and dilutions using manual sample treatments, assay conditions
which provided good sensitivity and low variance vitamin D
measurements were identified. In initial experiments, blood samples
were manually diluted 1:5 into a solution of 10 mg/mL pepsin in 100
mM citrate buffer pH 2.6, incubated for 5 minutes at 37.degree. C.
and then further diluted 1:10 into 50 mM TRIS pH 8.0 to neutralize
the sample and permanently inactivate the pepsin (providing a final
sample dilution of 1:50). Vitamin D controls were obtained from
Immunodiagnostic Systems, Inc. ("IDS", Scottsdale, Ariz. USA
85258). The dose response results obtained for the IDS serum-based
vitamin D calibrators and for the clinical samples were in good
agreement.
[0164] Vitamin D assay methods using pepsin degradation of vitamin
D binding proteins at low pH were then further optimized in
subsequent experiments, including experiments performed at an
operating temperature of 34.degree. C. It was determined that a
pepsin pre-incubation step was not required, as it was found that
the requisite digestion of carrier proteins took place within the
time (approximately 3 minutes) taken to mix the sample into the
extraction buffer. Thus, further assay conditions using pepsin were
determined which did not require extensive pre-incubation of the
sample. This sample treatment and assay protocol consisted of a 1:5
dilution of the sample into 10 mg/mL pepsin in 100 mM citrate
buffer pH 2.6, followed by a 1:8 dilution into 50 mM TRIS pH 8.0
which brought the pH of the diluted sample to 8.0 and neutralized
the pepsin. Then the sheep anti-25-hydroxy vitamin D antibody was
added and incubated with the sample for 10 minutes. Following
addition of the anti-25-hydroxy vitamin D antibody, the 25-hydroxy
vitamin D-AP conjugate was added, resulting in a final sample
dilution of 1:50. The mixture was then incubated on the anti-sheep
capture surface for 5 minutes, followed by a wash and 5 minute
substrate incubation.
[0165] The assay protocol steps were performed by automated device.
The following assay protocol was used:
[0166] 1. Sample was diluted 1:5 into 100 mM Citrate buffer pH 2.6
containing 10 mg/mL pepsin and mixed (final pH 3.9) for 5 minutes
at 34.degree. C.
[0167] 2. Treated sample was diluted a further 1:8 into 50 mM TRIS
buffer pH 8.0 to permanently deactivate the pepsin, and
anti-25-hydroxy vitamin D antibody in a blocking buffer was spiked
in and mixed. This mixture was incubated for 10 minutes.
[0168] 3. The AP-labeled 25-hydroxy vitamin D was added to the
sample mixture and then transferred to the capture surface
(biotinylated anti-sheep secondary antibody bound to
UltraAvidin.TM.-coated polystyrene) and incubated for 5
minutes.
[0169] 4. The surface was washed six times (1 minute for each wash
cycle) with 0.05% Tween20.RTM. in 50 mM TBS containing 0.05% sodium
azide at pH8.0.
[0170] 5. The washed capture surface was exposed to enzyme
substrate for five minutes and the chemiluminescence was read in an
automated luminometer.
[0171] Assays using anti-25-hydroxyvitamin D antibodies were able
to determine the amount of vitamin D (e.g., as 25-hydroxyvitamin D,
in both the D2 and D3 forms). The antibodies used had high affinity
for the 25-hydroxy vitamin D3-Alkaline Phosphate conjugate used in
the assay. As shown in the example shown in FIG. 4, sheep
monoclonal antibody Ab2F4 was able to detect 25-hydroxyvitamin D3
following pepsin treatment to release vitamin D from VDBPs.
[0172] FIG. 5 presents results of vitamin D measurements on
clinical (serum) samples obtained according to the methods of
Example 1, using a sheep anti-25-hydroxy vitamin D antibody
(AB5H10), shown compared to results obtained with a commercially
available method. The commercially available method used in this
example was the IDS EIA Kit.RTM. test (Immuno Diagnostic Systems
Ltd., Scottsdale, Ariz., USA), enzyme immunoassay. The results were
quite similar with both methods, confirming the usefulness of the
methods disclosed herein.
[0173] The Regression equation shown in FIG. 5 has the following
parameters:
y=0.81.times.(IDS result,ng/mL)+19.43;R=0.869;R.sup.2=0.7564
where "x" indicates multiplication.
Example 2: Balancing 25-hydroxy Vitamin D3 and 25-hydroxy Vitamin
D2 Reactivity
[0174] Assays using anti-25-hydroxyvitamin D antibodies were able
to determine the amount of vitamin D (as 25-hydroxy vitamin D, in
both the D2 and D3 forms). As shown in the example shown in FIG.
6A, sheep monoclonal antibody Ab2F4 was able to detect both
25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. Screening of
several sheep monoclonal antibodies reactive for both forms of
25-hydroxy vitamin D showed that Ab5H10 produced the best dose
response, but had higher affinity for 25-hydroxy vitamin D2 than
for 25-hydroxy vitamin D3, whereas Ab2F4 had higher reactivity for
25-hydroxy vitamin D3 than 25-hydroxy vitamin D2. Both antibodies
had high affinity for the 25-hydroxy vitamin D3-Alkaline Phosphate
conjugate used in the assay.
[0175] In order to achieve a more balanced reactivity for the
forms, the antibodies were combined in different proportions. This
experiment was performed with Sigma analytes diluted in low BSA
buffer (0.03% BSA in TBS). It was determined that a 2:1 ratio of
Ab5H10 to Ab2F4 produced the best results; a mixture of 100 ng/mL
Ab5H10 and 50 ng/mL Ab2F4 produced the best balance of reactivity
to 25-hydroxy vitamin D3 and 25-hydroxy vitamin D2, with 100%
reactivity to both forms in the clinical range. As shown in FIG.
6B, the 2:1 mixture of two sheep antibodies was able to detect both
25-hydroxy vitamin D2 and 25-hydroxy vitamin D3, with a combined
affinity that was substantially the same for both the D2 and D3
forms.
[0176] A set of 45 clinical samples was run to confirm the
performance of the final assay protocol. A calibration curve was
created in Low BSA buffer (0.03% BSA in TBS). As discussed above,
better results were obtained using balanced mixtures of
anti-vitamin D antibodies than were obtained using one
anti-25-hydroxy vitamin D antibody alone; the balance of reactivity
to 25-hydroxy vitamin D3 and 25-hydroxy vitamin D2 was optimized
using 2:1 ratios of anti-vitamin D antibodies Ab5H10 and Ab2F4.
[0177] The dose-response curve for such assays was determined which
provides a fit to the original data from clinical samples. The
dose-response curve of an exemplary experiment was fitted with the
following equation:
Conc=28.313.times.(((18944.016-1897.831)/(RLU-1897.831))-1){circumflex
over ( )}(1/2.597)
[0178] Where "x" indicates multiplication, and {circumflex over (
)} indicates exponentiation.
Example 3: Correlation of Assay Results with Those of Prior
Methods
[0179] Clinical samples were obtained which had a range of
25-hydroxy vitamin D concentrations; these samples were tested with
the IDS kit, with the Diasorin Liaison.RTM. methods, and with the
present methods as disclosed herein. FIG. 6 presents results of
25-hydroxy vitamin D measurements on clinical (serum) samples
obtained according to the methods of Example 1, where the
anti-25-hydroxy vitamin D antibody used in these measurements was a
2:1 mixture of Ab5H10 and Ab2F4 (final concentrations, 10 ng/mL
Ab5H10 and 5 ng/mL Ab2F4). These results are shown compared to
results obtained with the Diasorin Liaison.RTM. method, a test that
is widely used in the art. The Diasorin Liaison.RTM. test (DiaSorin
Corporation, Stillwater, Minn.) is a chemiluminescent assay in
which serum was incubated with antivitamin-D coated microparticles
and isoluminol derivative-conjugated 25-hydroxy vitamin D before
measurement of the chemiluminescent signal. Correlation of the
present methods with the Diasorin Liasion.RTM. assay results were
very good. A graph showing the results from the Diasorin
Liaison.RTM. assay and the results obtained by the methods
disclosed herein is presented in FIG. 7. The Regression equation
shown in FIG. 7 has the following parameters:
y=0.97.times.(Diasorin
Liaison.RTM.,ng/mL)+4.8;R=0.81;R.sup.2=0.664
where "x" indicates multiplication.
[0180] The results of the present methods applied to these samples
was also compared to results on these samples using the IDS EIA
Kit.RTM. test, an enzyme immunoassay available from Immuno
Diagnostic Systems Ltd., Scottsdale, Ariz., USA. Correlation of the
present methods with the IDS EIA kit results were also very good.
However, with respect to the IDS EIA results, one sample point was
considered an outlier, as the results for this point obtained using
the IDS kit appeared to be clearly different from the results
obtained by the Diasorin method and from the results obtained by
the present methods. This difference was possibly due to the
presence of high levels of 25-hydroxy Vitamin D2 in this sample and
to the fact that the IDS EIA kit reports only 75% of the D2 form of
vitamin D (see, e.g., Hollis, Clinical Chemistry 46(10):1657-1661
(2000) and Carter et al., Clinical Chemistry 50(11):2195-2197
(2004)), whereas the present methods and Diasorin systems fully
report the vitamin D2 content of samples. Thus, with the possible
exception of the one point mentioned above, the results were quite
similar with all methods, and the results of the present methods
correlate well with those of other tests used in the art. These
results confirm that the methods disclosed herein are suitable for
the measurement of vitamin D.
Example 4: Cross Reactivity
[0181] Tests were performed in order to determine whether
non-target compounds could interfere with the assay results. Such
tests, termed "cross-reactivity tests", measured the change in
measured 25-hydroxy vitamin D due to the presence of a non-target
substance by comparing the value of 25-hydroxy vitamin D measured
by the present methods where a potentially interfering compound was
in the sample, as compared to the value of 25-hydroxy vitamin D
measured by the present methods in the absence of the potentially
interfering compound. Cross reactivity testing was conducted using
the final assay conditions in a low BSA buffer matrix (0.03% BSA in
TBS). Cross-reactivity was reported in these assays as %
recovery=100.times.([measured vitamin D])/([nominal vitamin D]);
[measured vitamin D] is the 25-hydroxy vitamin D concentration
measured in the presence of the potentially interfering compound,
and [nominal vitamin D] is the 25-hydroxy vitamin D concentration
measured in the absence of the potentially interfering compound. In
general, a % recovery value between about 85% and about 115% does
not indicate significant cross-reactivity, while a % recovery value
greater than about 115% indicates cross-reactivity. (Note that an
interfering compound would displace labeled 25-hydroxy vitamin D in
the competition assays disclosed herein, so that the interfering
compound would cause an apparent (erroneous) increase in the
measured 25-hydroxy vitamin D.) For comparison, values for
25-hydroxy vitamin D3 (a direct target of the assay, and not a
cross-reactant) are presented in the table.
[0182] No cross reactivity was observed with vitamin D3
(Cholecalciferol), vitamin D2 (Ergocalciferol), or 24,25-dihydroxy
vitamin D3 as the potentially interfering compounds. Some cross
reactivity was observed with 1.alpha.,25-dihydroxy vitamin D3
(another hydroxylated metabolite of vitamin D) as the potentially
interfering compound. Such cross-reactivity does not present any
clinical concerns because blood levels of 1.alpha.,25-dihydroxy
vitamin D are in the pg/mL range, and so are on the order of
1000-fold lower than 25-hydroxy vitamin D, so that any possible
effects would be negligible. Cross reactivity with this metabolite
is common in 25-Hydroxyvitamin D assays.
[0183] Reported cross reactivity for various other 25-hydroxy
vitamin D methods are reported below for comparison. Table 1
reports cross-reactivity for various forms of vitamin D, over
several concentrations for each form of vitamin D, as measured by
the methods disclosed herein. For comparison, Table 2 reports
cross-reactivity results for various vitamin D assay methods,
including the present methods (right-most column).
TABLE-US-00001 TABLE 1 Cross Reactivity Conc., ng/mL [Test % Sub-
Signal, RLU Re- stance] CV CV cov- Test Substance ng/mL Mean % Mean
% ery 25-Hydroxy- 150 2432 11.9 104.5 20.9 70 vitamin D3 100 2941
2.7 76.5 3.4 77 50 4044 5.2 55.5 4.4 111 25 14076 9.4 20.8 13.0 83
10 20071 5.1 11.0 11.4 110 0 28281 2.8 4.2 9.3
1.alpha.,25-Dihydroxy- 150 2352 4.2 107.1 7.4 71 Vitamin D3 100
3642 3.0 60.6 2.8 61 50 7804 18.0 36.5 11.6 73 24,25-Dihydroxy- 150
22992 6.2 OORL Vitamin D3 100 23735 2.3 OORL 50 25059 3.2 OORL
Cholecalciferol 150 31607 9.8 OORL (Vitamin D3) 100 30864 2.4 OORL
50 31046 3.6 OORL Ergocalciferol ) 150 30935 4.0 OORL (Vitamin D2
100 27040 1.0 OORL 50 30040 3.9 OORL ("OORL" (out of range - low)
indicates that the analyte concentration was below the detection
limit)
TABLE-US-00002 TABLE 2 Reported Vitamin D Metabolite Cross
Reactivity for Various Methods DiaSorin DiaSorin 25- Liaison .RTM.
25 OH Roche Vitamin D Hydroxyvitamin Vitamin D Vitamin D3 IDS
Present Metabolite D 125I RIA TOTAL (25-OH) ELISA kit Methods
Vitamin D3 0.8% <1% <1% <0.01% <0.01% Vitamin D2 0.8%
<1% <1% <0.3% <0.01% 25(OH) D3 100% 100% 100% 100% 100%
25(OH) D2 100% 104% <10% 75% 110% 1,25(OH)2 D3 11% 17% up to
100% NR 70% 1,25(OH)2 D2 11% 40% NR NR NR 24,25(OH)2 D3 NR NR NR
>100% NR As stated in the manufacturer's product insert. NR =
Not Reported.
Example 5: Reagent Stability
[0184] The stability of pepsin in frozen aliquots of aqueous pepsin
solution and of dry, lyophilized pepsin (e.g., a lyophilized powder
formed from an aqueous pepsin solution) was measured. Pepsin was
lyophilized in containers suitable for use when reconstituted,
e.g., in an automated assay device. The pepsin was provided in a
solution at a concentration of 10 mg/mL and was lyophilized into
the containers to obtain the final reconstituted amount and
concentration equal to the concentration and amounts used in the
assay methods disclosed herein. The containers of lyophilized
reagent were placed into cartridges (arrays including several such
containers) and the reagent was reconstituted with 100 mM citrate
buffer (pH 2.6). Clinical samples were used for comparison, and the
vitamin D results were calculated for both conditions based on the
standard curve obtained for frozen pepsin/buffer aliquots. There
were no significant differences between the results obtained using
the lyophilized reagent and the results obtained using the (thawed)
frozen aliquot of pepsin reagent.
Example 6: Results Using Plasma, Whole Blood, Anticoagulants, and
Sample Dilution
[0185] Plasma: Many blood tests must be performed using blood
plasma, instead of serum, as results of some tests in serum may be
inaccurate or irregular. In order to determine whether or not
vitamin D assays using acidic pH and acid-active aspartyl
peptidases may be performed using blood plasma as well as blood
serum, assays using pepsin were performed in serum and in plasma.
In these experiments, matched blood samples were collected from 12
donors in lithium heparin tubes (to prevent coagulation of plasma)
and in serum tubes and centrifuged to obtain plasma and serum
respectively. The matched samples were tested according to the
methods disclosed herein and the results compared, based on the
assay buffer calibration curve. There was no significant difference
between serum and plasma results. Thus, the present vitamin D
assays may be performed using either serum or plasma. These results
are presented in FIG. 8.
[0186] Whole Blood: Whole blood must be treated to prepare plasma
or serum from a blood sample as obtained from a subject. Tests on
whole blood do not require such treatment, and thus may be simpler
than tests performed on plasma or serum. In order to determine
whether or not vitamin D assays using acidic pH and acid-active
aspartyl peptidases may be performed using whole blood, in addition
to using plasma or serum, vitamin D assays using pepsin were
performed in whole blood and the results compared with matched
results from plasma derived from the whole blood sample. In these
experiments, blood samples were collected from donors, and matched
whole blood and plasma samples were tested according to the methods
disclosed herein. As shown in FIG. 9, there was no significant
difference between whole blood and the plasma results. Thus, the
present vitamin D assays may be performed using either whole blood
or plasma. Together with the results shown in FIG. 8, the results
presented herein demonstrate that vitamin D may be measured in
whole blood, plasma, or serum according to the methods disclosed
herein.
[0187] Effect of Anticoagulant: Plasma prepared from blood is often
treated with either EDTA or lithium heparin as an anticoagulant in
the collection system. To test for any possible bias due to
anticoagulant, matched samples were collected from 10 donors in
EDTA and in lithium heparin tubes and centrifuged to prepare plasma
prior to analysis for vitamin D. There was no significant
difference between the lithium heparin and EDTA results.
[0188] Sample Dilution: To test for dilution linearity across the
range of vitamin D concentrations, a high concentration clinical
sample was diluted to provide a total of six samples, the high
concentration clinical sample and five dilutions of the high
concentration sample, for a total of six samples with vitamin D
concentrations of between about 14 ng/mL to about 108 ng/mL. The
nominal concentration of vitamin D was calculated and the recovered
concentration was compared to the calculated nominal concentration.
The recovery was excellent and was linear across the range.
[0189] The assays disclosed herein performed well for whole blood,
plasma and serum, and do not appear to be adversely affected by the
presence of anticoagulants or of sample dilution. No other test
method is believed to be able to make such vitamin D measurements
using whole blood samples.
[0190] While the above is a description of embodiments as described
herein, it is possible to use various alternatives, modifications
and equivalents.
[0191] This document contains material subject to copyright
protection. The copyright owner (Applicant herein) has no objection
to the facsimile reproduction by anyone of the patent document or
the patent disclosure, as they appear in the US Patent and
Trademark Office patent file or records, but otherwise reserves all
copyright rights whatsoever. The following notice shall apply:
Copyright 2013-2015 Theranos, Inc.
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