U.S. patent application number 16/225469 was filed with the patent office on 2019-05-02 for pretreatment agent in non-agglutination assays.
This patent application is currently assigned to Siemens Healthcare Diagnostics Inc.. The applicant listed for this patent is Siemens Healthcare Diagnostics Inc.. Invention is credited to Christy Schaible, Tie Q. Wei.
Application Number | 20190128877 16/225469 |
Document ID | / |
Family ID | 53371798 |
Filed Date | 2019-05-02 |
United States Patent
Application |
20190128877 |
Kind Code |
A1 |
Wei; Tie Q. ; et
al. |
May 2, 2019 |
PRETREATMENT AGENT IN NON-AGGLUTINATION ASSAYS
Abstract
Methods and reagents are disclosed for minimizing a false result
in an assay measurement for determining a concentration of an
analyte in a sample suspected of containing the analyte. The method
comprises pretreating both an antibody and a sample to be subjected
to a non-agglutination immunoassay. In the method the antibody and
the sample are combined with a pretreatment agent selected from the
group consisting of hydroxyphenyl-substituted C1-C5 carboxylic
acids and metallic salts thereof and halogen-substituted C1-C5
carboxylic acids and metallic salts thereof in an amount effective
to enhance the accuracy of the non-agglutination immunoassay.
Inventors: |
Wei; Tie Q.; (Wilmington,
DE) ; Schaible; Christy; (Oxford, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Healthcare Diagnostics Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Siemens Healthcare Diagnostics
Inc.
Tarrytown
NY
|
Family ID: |
53371798 |
Appl. No.: |
16/225469 |
Filed: |
December 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15103534 |
Jun 10, 2016 |
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PCT/US2014/069520 |
Dec 10, 2014 |
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16225469 |
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61915755 |
Dec 13, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5306 20130101;
G01N 33/9493 20130101 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/94 20060101 G01N033/94 |
Claims
1.-11. (canceled)
12. A method of determining immunosuppressant drug in a sample
suspected of containing the immunosuppressant drug, the method
comprising a non-agglutination immunoassay comprising: (a)
providing in combination in a medium: (i) the sample, (ii) a
releasing agent for releasing the immunosuppressant drug from
endogenous binding substances, wherein the releasing agent is
selected from the group consisting of one or more of a structural
analog of the immunosuppressant drug and a hemolytic agent, (iii)
an antibody reagent comprising an antibody for the
immunosuppressant drug, (iv) a pretreatment agent selected from the
group consisting of hydroxyphenyl-substituted C1-C5 carboxylic
acids and metallic salts thereof and halogen-substituted C1-C5
carboxylic acids and metallic salts thereof in an amount effective
to enhance the accuracy of the non-agglutination immunoassay; (b)
incubating the medium under conditions for binding of the antibody
for the immunosuppressant drug to the immunosuppressant drug
suspected of being in the sample, wherein the incubation
temperature is 5 degrees C. to 99 degrees C. and the time period
for the incubation is 0.2 seconds to 24 hours, (c) examining the
medium for the presence of a complex comprising the
immunosuppressant drug and the antibody for the immunosuppressant
drug, the presence and/or amount of the complex indicating the
presence and/or amount of the immunosuppressant drug in the sample,
wherein the immunosuppressant drug is selected from the group
consisting of cyclosporine, everolimus, siroliumus and
tacrolimus.
13. The method according to claim 12 wherein the pretreatment agent
is a metallic salt of salicylic acid.
14. The method according to claim 12 wherein the pretreatment agent
is a metallic salt of a chloro-substituted C1-C5 carboxylic
acid.
15. The method according to claim 12 wherein the pretreatment agent
is a metallic salt of a chloro-substituted acetic acid.
16. The method according to claim 12 wherein the pretreatment agent
is a metallic salt of trichloroacetic acid.
17. The method according to claim 12 wherein the antibody reagent
further comprises a label.
18. The method according to claim 12 wherein the combination
further comprises a particle.
19. The method according to claim 12 wherein the antibody reagent
further comprises an enzyme label and the combination further
comprises a magnetic particle.
20. The method according to claim 12 wherein the combination
further comprises structural analog of the immunosuppressant drug
and at least one of the antibody for the immunosuppressant drug or
the analog comprises a label.
21. The method according to claim 12, wherein the releasing agent
further comprises a solubility agent.
Description
BACKGROUND
[0001] The invention relates to methods and kits for determining
the concentration of an analyte in a sample suspected of containing
the analyte. More particularly, the invention relates to reducing
false results in the measurements conducted during the above
methods for determining the concentration of an analyte in a
sample.
[0002] The body relies upon a complex immune response system to
distinguish self from non-self. At times, the body's immune system
must be controlled in order to either augment a deficient response
or suppress an excessive response. For example, when organs such as
kidney, heart, heart-lung, bone marrow and liver are transplanted
in humans, the body will often reject the transplanted tissue by a
process referred to as allograft rejection.
[0003] In treating allograft rejection, the immune system is
frequently suppressed in a controlled manner with drug therapy.
Immunosuppressant drugs are carefully administered to transplant
recipients in order to help prevent allograft rejection of non-self
tissue. Two most commonly administered immunosuppressive drugs to
prevent organ rejection in transplant patients are Cyclosporine
(CSA) and FK-506 (FK or tacrolimus). Another drug that finds use as
an immunosuppressant in the United States and other countries is
sirolimus, also known as rapamycin. Derivatives of sirolimus are
also said to be useful as immunosuppressants. Such derivatives
include, for example, Everolimus.
[0004] The side effects associated with some immunosuppressant
drugs can be controlled in part by carefully controlling the level
of the drug present in a patient. Therapeutic monitoring of
concentrations of immunosuppressant drugs and related drugs in
blood is required to optimize dosing regimes to ensure maximal
immunosuppression with minimal toxicity. Although immunosuppressant
drugs are highly effective immunosuppressive agents, their use must
be carefully managed because the effective dose range is often
narrow and excessive dosage can result in serious side effects. On
the other hand, too little dosage of an immunosuppressant can lead
to tissue rejection. Because the distribution and metabolism of an
immunosuppressant drug can vary greatly between patients and
because of the wide range and severity of adverse reactions,
accurate monitoring of the drug level is essential.
[0005] In the therapeutic drug monitoring field, selectively
detecting a drug over other substances in a sample suspected of
containing the drug is often an important goal for designing
immunoassays. This is especially true for immunosuppressant drugs.
For that reason, HPLC tandem MS assays have become standard methods
for the measurement of sirolimus, tacrolimus and other
immunosuppressant drugs due to their ability to selectively measure
the parent drug. However, the above methods are costly and
time-consuming and are often employed to verify positive results
obtained by another assay method first rather than being used in
laboratories as an initial determination.
[0006] Most whole blood assays for immunosuppressant drugs require
a manual step using reagents to extract the drug from blood
constituents. As a result, the drug molecules and drug metabolite
molecules are dissociated from endogenous binding proteins and are
extracted into a relatively clean solution in which plasma proteins
and lipoprotein particles as well as most other molecules are
removed. Because precipitation techniques are usually used, the
extracted sample is basically free of most blood macromolecules
including drug-binding proteins. Thus, in the extracted samples,
the parent drug and its metabolites are dissolved as unbound,
individual molecules and compete with one another for reaction with
an assay antibody in the immunoreaction mixture. The binding of the
assay antibody to the drug occurs in the absence of most endogenous
substances in these assays. The cross-reactivity of a drug
metabolite depends mostly on its antibody binding affinity in such
assays.
[0007] In a homogeneous assay for an immunosuppressant drug where
there is no manual extraction or separation of the drug from blood
constituents, an antibody for the immunosuppressant drug has to
detect the drug in the presence of most or all blood constituents.
The presence of these constituents might interfere with the binding
of the antibody to the immunosuppressant drug and lead to a false
assay result.
[0008] There is, therefore, a continuing need to develop fast and
accurate diagnostic methods to measure levels of analytes in
samples taken from a patient. The methods should be fully
automatable and be accurate even when conducted on samples having
various interfering substances present. The assay should provide an
accurate measurement of the amount of the analyte in the sample,
while minimizing inaccuracies resulting from interfering substances
present in the sample. Reduction in false assay measurements is
important to the accuracy of the methods.
SUMMARY
[0009] One example in accordance with the principles described
herein is directed to a method of pretreating both an antibody and
a sample to be subjected to a non-agglutination immunoassay. In the
method the antibody and the sample are combined with a pretreatment
agent selected from the group consisting of
hydroxyphenyl-substituted C1-C5 carboxylic acids and metallic salts
thereof and halogen-substituted C1-C5 carboxylic acids and metallic
salts thereof in an amount effective to enhance the accuracy of the
non-agglutination immunoassay.
[0010] Another example in accordance with the principles described
herein is directed to a method of determining an analyte in a
sample suspected of containing the analyte. The method comprises a
non-agglutination immunoassay. In the non-agglutination
immunoassay, a combination is provided in a medium where the
combination comprises the sample, an antibody reagent comprising an
antibody for the analyte, and a pretreatment agent selected from
the group consisting of hydroxyphenyl-substituted C1-C5 carboxylic
acids and metallic salts thereof and halogen-substituted C1-C5
carboxylic acids and metallic salts thereof in an amount effective
to enhance the accuracy of the non-agglutination immunoassay. The
medium is incubated under conditions for binding of the antibody
for the analyte to analyte suspected of being in the sample. The
medium is examined for the presence of a complex comprising the
analyte and the antibody for the analyte, the presence and/or
amount of the complex indicating the presence and/or amount of the
analyte in the sample.
[0011] Another example in accordance with the principles described
herein is directed to a method of determining an immunosuppressant
drug in a sample suspected of containing the immunosuppressant
drug. The method comprising a non-agglutination immunoassay. In the
method a combination is provided in a medium. The combination
comprises the sample, a releasing agent for releasing the
immunosuppressant drug from endogenous binding substances, an
antibody reagent comprising an antibody for the immunosuppressant
drug, and a pretreatment agent selected from the group consisting
of hydroxyphenyl-substituted C1-C5 carboxylic acids and metallic
salts thereof and halogen-substituted C1-C5 carboxylic acids and
metallic salts thereof in an amount effective to enhance the
accuracy of the non-agglutination immunoassay. The medium is
incubated under conditions for binding of the antibody for the
immunosuppressant drug to the immunosuppressant drug suspected of
being in the sample. The medium is examined for the presence of a
complex comprising the immunosuppressant drug and the antibody for
the immunosuppressant drug, the presence and/or amount of the
complex indicating the presence and/or amount of the
immunosuppressant drug in the sample.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
General Discussion
[0012] The present invention is directed to the accurate
measurement of analyte concentration in a sample by reducing or
eliminating false assay results in a non-agglutination immunoassay.
In examples of methods in accordance with the principles described
herein, an antibody and a sample to be subjected to a
non-agglutination immunoassay are combined with a pretreatment
agent selected from the group consisting of
hydroxyphenyl-substituted C1-C5 carboxylic acids and metallic salts
thereof and halogen-substituted C1-C5 carboxylic acids and metallic
salts thereof, or a combination of two or more of the above, in an
amount effective to enhance the accuracy of the non-agglutination
immunoassay.
[0013] An "antibody" is a member of a specific binding pair ("sbp
member"), which is one of two different molecules, having an area
on the surface or in a cavity, which specifically binds to and is
thereby defined as complementary with a particular spatial and
polar organization of the other molecule. The members of the
specific binding pair will usually be members of an immunological
pair such as antigen-antibody, although other specific binding
pairs such as, for example, biotin-avidin, hormones-hormone
receptors, enzyme-substrate, nucleic acid duplexes, IgG-protein A,
and polynucleotide pairs such as DNA-DNA, DNA-RNA, are not
immunological pairs but are included within the scope of sbp
member. Accordingly, specific binding involves the specific
recognition of one of two different molecules for the other
compared to substantially less recognition of other molecules. On
the other hand, non-specific binding involves non-covalent binding
between molecules that is relatively independent of specific
surface structures. Non-specific binding may result from several
factors including hydrophobic interactions between molecules.
[0014] A "non-agglutination" immunoassay is an assay employing at
least one antibody or similar macromolecular member of a specific
binding pair where the assay does not involve the aggregation of
substances such as particulate materials, for example, bacteria,
cells, or latex particles, by virtue of the binding of antigens on
the substances to the antibody or similar macromolecular sbp
member.
[0015] C1-C5 carboxylic acids refers to carboxylic acids having
from 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon
atoms, or 1 to 2 carbon atoms, or 2 to 5 carbon atoms, or 2 to 4
carbon atoms, or 2 to 3 carbon atoms, or 3 to 5 carbon atoms, or 4
to 5 carbon atoms. Examples of C1-C5 carboxylic acids include, by
way of illustration and not limitation, formic acid, acetic acid,
propionic acid, n-butanoic acid, iso-butanoic acid, n-pentanoic
acid, iso-pentanoic acid, for example.
[0016] In some examples, the C1-C5 carboxylic acid is substituted
with a hydroxyphenyl substituent, which is a phenyl group
comprising at least one hydroxy group, or at least two hydroxy
groups. The hydroxy substituent may be ortho (o), meta (m) or para
(p) to the carbon atom of the phenyl ring that is attached to the
C1-C5 carboxylic acid. Examples of hydroxyphenyl C1-C5 carboxylic
acids include, but are not limited to, (o-hydroxyphenyl)formic acid
(salicylic acid), (m-hydroxyphenyl)formic acid,
(p-hydroxyphenyl)formic acid, (o-hydroxyphenyl)acetic acid,
(m-hydroxyphenyl)acetic acid, and (p-hydroxyphenyl)acetic acid, for
example.
[0017] In some examples, the hydroxyphenyl-substituted C1-C5
carboxylic acids are in the form of a metal salt. The metal of the
metal salt may be, by way of illustration and not limitation, a
metal of Group 1 of the periodic table, or a metal of Group 2 of
the periodic table, for example. In some examples, the metal of the
metallic salt of hydroxyphenyl-substituted C1-C5 carboxylic acid
is, but is not limited to, sodium, potassium, or lithium, for
example. In a particular example in accordance with the principles
described herein, the metallic salt of hydroxyphenyl-substituted
C1-C5 carboxylic acid is sodium salicylate.
[0018] Halogen-substituted C1-C5 carboxylic acids are those C1-C5
carboxylic acids that comprise at least one halogen substituent, or
at least two halogen substituents, or at least 3 halogen
substituents. In some examples, the number of halogen substituents
may be in the range of, for example, 1-4, or 1-3, or 1-2, or 2-4,
or 3-4, or 2-3. In some examples, the number of halogen
substituents may be 1, or 2, or 3, or 4, for example. The halogen
substituent may be, for example, one or more of chlorine, bromine,
fluorine and iodine. In some examples in accordance with the
principles described herein, the halogen-substituted C1-C5
carboxylic acid may be, but is not limited to, chloroacetic acid,
dichloroacetic acid, trichloroacetic acid, 2-chloropropionic acid,
2,2-dichloropropionic acid, 2,2,2-trichloropropionic acid,
2,3-dichloropropionic acid, 2,3,3-trichloropropionic acid,
bromoacetic acid, dibromoacetic acid, tribromoacetic acid,
2-bromopropionic acid, 2,2-dibromopropionic acid,
2,2,2-tribromopropionic acid, 2,3-dibromopropionic acid,
2,3,3-tribromopropionic acid, iodoacetic acid, diiodoacetic acid,
triiodoacetic acid, 2-iodopropionic acid, 2,2-diiodopropionic acid,
2,2,2-triiodopropionic acid, 2,3-diiodopropionic acid, and
2,3,3-triiodopropionic acid, for example. In some examples, the
halogen-substituted C1-C5 carboxylic acid may be in the form of a
metallic salt where the metal of the metallic salt is from Group 1
or Group 2 of the periodic table as discussed above for the
metallic salt of the hydroxyphenyl-substituted C1-C5 carboxylic
acids. In a particular example in accordance with the principles
described herein, the metallic salt of halogen-substituted C1-C5
carboxylic acid is sodium trichloroacetate.
[0019] In examples of methods in accordance with the principles
described herein, an antibody and a sample to be subjected to a
non-agglutination immunoassay are combined with a pretreatment
agent as discussed above in an aqueous medium, which may be an
aqueous buffered medium. The medium may be a medium in which a
non-agglutination immunoassay is conducted in accordance with the
principles described herein or a medium employed for conducting the
pretreatment method. The pH for the aqueous medium will usually be
in the range of about 4 to about 11, or in the range of about 5 to
about 10, or in the range of about 6.5 to about 9.5.
[0020] Various buffers may be used to achieve the desired pH and
maintain the pH during the procedure. Illustrative buffers include
borate, phosphate, carbonate, tris, barbital and the like. The
particular buffer employed is not critical, but in an individual
assay one or another buffer may be preferred. Various ancillary
materials may be employed in the above methods. For example, in
addition to buffers the medium may comprise stabilizers for the
medium and for the reagents employed. In some embodiments, in
addition to these additives, proteins may be included, such as
albumins; quaternary ammonium salts; polyanions such as dextran
sulfate; and binding enhancers, for example. All of the above
materials are present in a concentration or amount sufficient to
achieve the desired effect or function.
[0021] The pretreatment agent is employed in an amount sufficient
to enhance the accuracy of the non-agglutination assay. The amount
of the pretreatment agent is dependent on one or more of the nature
of the non-agglutination assay, the nature of the antibody reagent,
the amount of the sample, the suspected range of the amount of an
analyte in a sample, the amount of other substances suspected of
being in the sample, and the presence of metabolites and/or
phospholipids, for example. In some examples, the amount or
concentration (weight percent) of the pretreatment agent in the
assay medium is about 1% to about 75%, or about 1% to about 50%, or
about 1% to about 40%, or about 1% to about 30%, or about 1% to
about 20%, or about 1% to about 10%, or about 1% to about 5%, or
about 5% to about 75%, or about 5% to about 50%, or about 5% to
about 40%, or about 5% to about 30%, or about 5% to about 20%, or
about 5% to about 10%, or about 10% to about 75%, or about 10% to
about 50%, or about 10% to about 40%, or about 10% to about 30%, or
about 10% to about 20%, or about 10% to about 15%, for example.
[0022] The sample to be tested may be non-biological or biological.
"Non-biological samples" are those that do not relate to a
biological material and include, for example, soil samples, water
samples, air samples, samples of other gases and mineral samples.
The phrase "biological sample" refers to any biological material
such as, for example, body fluid, body tissue, body compounds and
culture media. The sample may be a solid, semi-solid or a fluid (a
liquid or a gas) from any source. In some embodiments the sample
may be a body excretion, a body aspirant, a body excisant or a body
extractant. The body is usually that of a mammal and in some
embodiments the body is a human body. Body excretions are those
substances that are excreted from a body (although they also may be
obtained by excision or extraction) such as, for example, urine,
feces, stool, vaginal mucus, semen, tears, breath, sweat, blister
fluid and inflammatory exudates. Body excisants are those materials
that are excised from a body such as, for example, skin, hair and
tissue samples including biopsies from organs and other body parts.
Body aspirants are those materials that are aspirated from a body
such as, for example, mucus, saliva and sputum. Body extractants
are those materials that are extracted from a body such as, for
example, whole blood, plasma, serum, spinal fluid, cerebral spinal
fluid, lymphatic fluid, synovial fluid and peritoneal fluid.
[0023] The amount of the sample subjected to an assay is dependent
on one or more of the nature of the analyte, the nature of the
assay, the nature of the various reagents for conducting the assay,
and the nature of the complex comprising the analyte, for example.
In some examples, the volume of the sample is about 1 .mu.L to
about 100 .mu.L, or about 2 .mu.L to about 100 .mu.L, or about 5
.mu.L to about 100 .mu.L, or about 10 .mu.L to about 100 .mu.L, or
about 1 .mu.L to about 80 .mu.L, or about 1 .mu.L to about 60
.mu.L, or about 1 .mu.L to about 40 .mu.L, or about 1 .mu.L to
about 20 .mu.L, or about 5 .mu.L to about 50 .mu.L, or about 10
.mu.L to about 50 .mu.L, for example.
[0024] The analyte is a substance of interest or the compound or
composition to be detected and/or quantitated. Analytes include, by
way of illustration and not limitation, therapeutic drugs, drugs of
abuse, metabolites, pesticides, volatile organic compounds,
semi-volatile organic compounds, non-volatile organic compounds,
proteins, polysaccharides, pollutants, toxins, lipids and nucleic
acids, (DNA, RNA), for example.
[0025] Representative drug analytes, by way of illustration and not
limitation, include alkaloids, steroids, lactams,
aminoalkylbenzenes, benzheterocyclics, purines, drugs derived from
marijuana, hormones, polypeptides which includes proteins,
immunosuppressants, vitamins, prostaglandins, tricyclic
antidepressants, anti-neoplastics, nucleosides and nucleotides
including polynucleosides and polynucleotides, miscellaneous
individual drugs which include methadone, meprobamate, serotonin,
meperidine, lidocaine, procainamide, acetylprocainamide,
propranolol, griseofulvin, valproic acid, butyrophenones,
antihistamines, chloramphenicol, anticholinergic drugs, and
metabolites and derivatives of all of the above.
[0026] Also included within the term analyte are metabolites
related to disease states, aminoglycosides, such as gentamicin,
kanamicin, tobramycin, and amikacin, and pesticides such as, for
example, polyhalogenated biphenyls, phosphate esters,
thiophosphates, carbamates and polyhalogenated sulfenamides and
their metabolites and derivatives.
[0027] The term analyte also includes combinations of two or more
of polypeptides and proteins, polysaccharides and nucleic acids.
Such combinations include, for example, components of bacteria,
viruses, chromosomes, genes, mitochondria, nuclei and cell
membranes. Protein analytes include, for example, immunoglobulins,
cytokines, enzymes, hormones, cancer antigens, nutritional markers
and tissue specific antigens. Such proteins include, by way of
illustration and not limitation, protamines, histones, albumins,
globulins, scleroproteins, phosphoproteins, mucoproteins,
chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cell
receptors, proteoglycans, HLA, unclassified proteins, e.g.,
somatotropin, prolactin, insulin, pepsin, proteins found in human
plasma, blood clotting factors, protein hormones such as, e.g.,
follicle-stimulating hormone, luteinizing hormone, luteotropin,
prolactin, chorionic gonadotropin, tissue hormones, cytokines,
cancer antigens such as, e.g., PSA, CEA, a-fetoprotein, acid
phosphatase, CA19.9 and CA125, tissue specific antigens, such as,
e.g., alkaline phosphatase, myoglobin, CPK-MB and calcitonin, and
peptide hormones. Other polymeric materials of interest are
mucopolysaccharides and polysaccharides. As indicated above, the
term analyte further includes oligonucleotide and polynucleotide
analytes such as m-RNA, r-RNA, t-RNA, DNA and DNA-RNA duplexes, for
example.
[0028] In some examples in accordance with the principles described
herein, the analyte is an immunosuppressant drug, which is a
therapeutic drug that is administered to transplant recipients in
order to help prevent allograft rejection of non-self tissue.
Immunosuppressant drugs can be classified as follows:
glucocorticoids, cytostatics, antibodies, drugs acting on
immunophilins and other drugs such as interferons, opiates INF
binding proteins, mycophenolate, and FTY720, for example. A
particular class of immunosuppressant drugs comprises those drugs
that act on immunophilins. Immunophilins are an example of
high-affinity, specific binding proteins having physiological
significance. Two distinct families of immunophilins are presently
known: cyclophilins and macrophilins, the latter of which
specifically bind, for example, tacrolimus or sirolimus. The
immunosuppressant drugs that act on immunophilin include, for
example, cyclosporin (including cyclosporin A, cyclosporin B,
cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F,
cyclosporin G, cyclosporin H, cyclosporin I), tacrolimus (FK506,
PROGRAF.RTM.), sirolimus (rapamycin, RAPAMUNE.RTM.), and everolimus
(RAD, CERTICAN.RTM.).
[0029] Some examples in accordance with the principles described
herein are directed to non-agglutination assay methods of
determining an analyte in a sample suspected of containing the
analyte. In the non-agglutination assay, a combination is provided
in a medium where the combination comprises the sample, an antibody
reagent comprising an antibody for the analyte, and an agent
selected from the group consisting of metallic salts of
hydroxyphenyl-substituted C1-C5 carboxylic acids and
halogen-substituted C1-C5 carboxylic acids in an amount effective
to enhance the accuracy of the non-agglutination assay.
[0030] The antibody reagent comprises an antibody for the analyte,
that is, an antibody that specifically binds to the analyte. The
antibody reagent may further comprise one or more labels or labeled
reagent. The antibody reagent includes those agents necessary for
the detection of the analyte. The label is usually part of a signal
producing system ("sps"). The nature of the label is dependent on
the particular non-agglutination assay format. An sps usually
includes one or more components, at least one component being a
detectable label, which generates a detectable signal that relates
to the amount of bound and/or unbound label, i.e. the amount of
label bound or not bound to the analyte being detected or to an
agent that reflects the amount of the analyte to be detected. The
label is any molecule that produces or can be induced to produce a
signal, and may be, for example, a fluorescer, radiolabel, enzyme,
chemiluminescer or photosensitizer. Thus, the signal is detected
and/or measured by detecting enzyme activity, luminescence, light
absorbance or radioactivity, and so forth, as the case may be.
[0031] Suitable labels include, by way of illustration and not
limitation, enzymes such as 3-galactosidase, alkaline phosphatase,
glucose-6-phosphate dehydrogenase ("G6PDH") and horseradish
peroxidase; ribozyme; a substrate for a replicase such as QB
replicase; promoters; dyes; fluorescers, such as fluorescein,
isothiocyanate, rhodamine compounds, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, and fluorescamine; complexes such
as those prepared from CdSe and ZnS present in semiconductor
nanocrystals known as Quantum dots; chemiluminescers such as
isoluminol and acridinium esters, for example; sensitizers;
coenzymes; enzyme substrates; radiolabels such as .sup.125I,
.sup.131I, .sup.14C, .sup.3H, .sup.57Co and .sup.75Se; particles
such as latex particles, carbon particles, metal particles
including magnetic particles, e.g., chromium dioxide (CrO.sub.2)
particles, and the like; metal sol; crystallite; liposomes; cells,
etc., which may be further labeled with a dye, catalyst or other
detectable group. Suitable enzymes and coenzymes are disclosed in
Litman, et al., U.S. Pat. No. 4,275,149, columns 19-28, and
Boguslaski, et al., U.S. Pat. No. 4,318,980, columns 10-14;
suitable fluorescers and chemiluminescers are disclosed in Litman,
et al., U.S. Pat. No. 4,275,149, at columns 30 and 31; which are
incorporated herein by reference.
[0032] The label can directly produce a signal and, therefore,
additional components are not required to produce a signal.
Numerous organic molecules, for example fluorescers, are able to
absorb ultraviolet and visible light, where the light absorption
transfers energy to these molecules and elevates them to an excited
energy state. This absorbed energy is then dissipated by emission
of light at a second wavelength. Other labels that directly produce
a signal include radioactive isotopes and dyes.
[0033] Alternately, the label may need other components to produce
a signal, and the signal producing system would then include all
the components required to produce a measurable signal. Such other
components may include substrates, coenzymes, enhancers, additional
enzymes, substances that react with enzymic products, catalysts,
activators, cofactors, inhibitors, scavengers, metal ions, and a
specific binding substance required for binding of signal
generating substances. A detailed discussion of suitable signal
producing systems can be found in Ullman, et al., U.S. Pat. No.
5,185,243, columns 11-13, incorporated herein by reference.
[0034] The label or other sps members can be bound to a support. An
analyte derivative or analyte analog may be bound to a solid
support in any manner known in the art, provided only that the
binding does not substantially interfere with the analogs ability
to bind with an antibody. In some embodiments, the analyte
derivative or analyte analog may be coated or covalently bound
directly to the solid phase or may have layers of one or more
carrier molecules such as poly(amino acids) including proteins such
as serum albumins or immunoglobulins, or polysaccharides
(carbohydrates) such as, for example, dextran or dextran
derivatives. Linking groups may also be used to covalently couple
the solid support and the analyte analog. Other methods of binding
the analyte derivatives are also possible. For instance, a solid
support may have a coating of a binder for a small molecule such
as, for example, avidin or an antibody, and a small molecule such
as, e.g., biotin or hapten, can be bound to the analyte derivative
or vice versa. The binding of components to the surface of a
support may be direct or indirect, covalent or non-covalent and can
be accomplished by well-known techniques, commonly available in the
literature. See, for example, "Immobilized Enzymes," Ichiro
Chibata, Halsted Press, New York (1978) and Cautrecasas, J. Biol.
Chem., 245:3059 (1970).
[0035] The support may be comprised of an organic or inorganic,
solid or fluid, water insoluble material, which may be transparent
or partially transparent. The support can have any of a number of
shapes, such as particle, including bead, film, membrane, tube,
well, strip, rod, planar surfaces such as, e.g., plate, paper,
etc., or fiber. Depending on the type of assay, the support may or
may not be suspendable in the medium in which it is employed.
Examples of suspendable supports are polymeric materials such as,
but not limited to, latex, lipid bilayers or liposomes, oil
droplets, cells and hydrogels, and magnetic particles, for example.
Other support compositions include polymers, such as
nitrocellulose, cellulose acetate, poly (vinyl chloride),
polyacrylamide, polyacrylate, polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, and poly(vinyl butyrate), for example, which
can be either used by themselves or in conjunction with other
materials.
[0036] The support may be a particle. The particles should have an
average diameter of at least about 0.02 microns and not more than
about 100 microns. In some embodiments, the particles have an
average diameter from about 0.05 microns to about 20 microns, or
from about 0.3 microns to about 10 microns. The particle may be
organic or inorganic, swellable or non-swellable, porous or
non-porous, preferably of a density approximating water, generally
from about 0.7 g/mL to about 1.5 g/mL, and composed of material
that can be transparent, partially transparent, or opaque. The
particles can be biological materials such as cells and
microorganisms, e.g., erythrocytes, leukocytes, lymphocytes,
hybridomas, streptococcus, Staphylococcus aureus, or E. coli,
viruses, for example. The particles can also be particles comprised
of organic and inorganic polymers, liposomes, latex particles,
magnetic or non-magnetic particles, phospholipid vesicles,
chylomicrons, or lipoproteins, for example. In some examples, the
particles are chromium dioxide (chrome) particles or latex
particles.
[0037] The polymer particles can be formed of addition or
condensation polymers. The particles will be readily dispersible in
an aqueous medium and can be adsorptive or functionalizable so as
to permit conjugation to an analyte analog, either directly or
indirectly through a linking group. The particles can also be
derived from naturally occurring materials, naturally occurring
materials that are synthetically modified, and synthetic materials.
Among organic polymers of particular interest are polysaccharides,
particularly cross-linked polysaccharides, such a agarose, which is
available as SEPHAROSE.RTM., dextran, available as SEPHADEX.RTM.
and SEPHACRYL.RTM., cellulose, and starch, for example; addition
polymers, such as polystyrene, polyvinyl alcohol, homopolymers and
copolymers of derivatives of acrylate and methacrylate,
particularly esters and amides having free hydroxyl
functionalities, for example.
[0038] The label and/or other sps member may be bound to an sbp
member or another molecule. For example, the label can be bound
covalently to an sbp member such as, for example, an antibody, a
receptor for an antibody, a receptor that is capable of binding to
a small molecule conjugated to an antibody, or a ligand (analyte)
analog. Bonding of the label to the sbp member may be accomplished
by chemical reactions that result in replacing a hydrogen atom of
the label with a bond to the sbp member or may include a linking
group between the label and the sbp member. Other sps members may
also be bound covalently to sbp members. For example, two sps
members such as a fluorescer and quencher can each be bound to a
different antibody where the antibodies form a specific complex
with the analyte. Formation of the complex brings the fluorescer
and quencher in close proximity, thus permitting the quencher to
interact with the fluorescer to produce a signal. Methods of
conjugation are well known in the art. See, for example,
Rubenstein, et al., U.S. Pat. No. 3,817,837, incorporated herein by
reference.
[0039] Enzymes of particular interest as label proteins are redox
enzymes, particularly dehydrogenases such as glucose-6-phosphate
dehydrogenase and lactate dehydrogenase, for example, and enzymes
that involve the production of hydrogen peroxide and the use of the
hydrogen peroxide to oxidize a dye precursor to a dye. Particular
combinations include saccharide oxidases, e.g., glucose and
galactose oxidase, or heterocyclic oxidases, such as uricase and
xanthine oxidase, coupled with an enzyme that employs the hydrogen
peroxide to oxidize a dye precursor, that is, a peroxidase such as
horse radish peroxidase, lactoperoxidase, or microperoxidase.
Additional enzyme combinations are known in the art. When a single
enzyme is used as a label, other enzymes may find use such as
hydrolases, transferases, and oxidoreductases, preferably
hydrolases such as alkaline phosphatase and .beta.-galactosidase.
Alternatively, luciferases may be used such as firefly luciferase
and bacterial luciferase.
[0040] Illustrative co-enzymes that find use include NAD[H],
NADP[H], pyridoxal phosphate, FAD[H], FMN[H], for example, usually
coenzymes involving cycling reactions. See, for example, U.S. Pat.
No. 4,318,980, the disclosure of which is incorporated herein by
reference.
[0041] With label proteins such as, for example, enzymes, the
weight average molecular weight range will be from about 10,000 to
about 600,000, or from about 10,000 to about 300,000 molecular
weight. There is usually at least about 1 analyte analog per about
200,000 molecular weight, or at least about 1 per about 150,000
molecular weight, or at least about 1 per about 100,000 molecular
weight, or at least about 1 per about 50,000 molecular weight, for
example. In the case of enzymes, the number of analyte analog
groups is from 1 to about 20, about 2 to about 15, about 3 to about
12, or about 6 to about 10, for example.
[0042] The term "non-poly(amino acid) labels" includes those labels
that are not proteins (e.g., enzymes). The non-poly(amino acid)
label is capable of being detected directly or is detectable
through a specific binding reaction that produces a detectable
signal. The non-poly(amino acid) labels include, for example,
radioisotopes, luminescent compounds, supports, e.g., particles,
plates, beads, etc., polynucleotides, and the like. More
particularly, the non-poly(amino acid) label can be isotopic or
non-isotopic, usually non-isotopic, and can be a polynucleotide
coding for a catalyst, promoter, dye, coenzyme, enzyme substrate,
radioactive group, a small organic molecule (including, e.g.,
biotin, fluorescent molecules, chemiluminescent molecules, and the
like), amplifiable polynucleotide sequence, a support such as, for
example, a particle such as latex or carbon particle or chromium
dioxide (chrome) particle, metal sol, crystallite, liposome, cell,
which may or may not be further labeled with a dye, catalyst or
other detectable group, for example.
[0043] In some examples in accordance with the principles described
herein such as, for example, an immunosuppressant drug analyte, a
releasing agent may be employed either prior to combining the
sample with the antibody reagent or in the combination of the
antibody reagent, the sample and the pretreatment agent.
[0044] The releasing agent displaces the analyte from endogenous
binding moieties. In some embodiments the releasing agent has high
binding affinity to the endogenous binding proteins so that it
readily displaces the analyte, and its metabolites in some
instances, from endogenous binding proteins. In addition, the
releasing agent does not bind to any significant degree to an
antibody for the analyte that is used in the assay. By the phrase
"does not bind to any significant degree" is meant that the extent
of binding should be low enough so that an accurate assay for the
analyte may be carried out. The releasing agent may be any moiety,
either a single compound or a mixture of compounds, which
accomplishes the desired result of displacement with no significant
binding to an assay antibody.
[0045] In some examples the releasing agent is an analog, including
structural analogs, of the analyte. A releasing agent that is an
analyte analog is a modified drug that can displace the analogous
analyte from a binding protein but does not compete to any
substantial degree for a receptor such as an antibody for the
analyte. The modification provides means to join an analyte analog
to another molecule. The analyte analog will usually differ from
the analyte by more than replacement of a hydrogen with a bond
which links the analyte analog to a hub or label, but need not. The
analyte analog may be, for example, the analyte conjugated to
another molecule through a linking group, for example. For analytes
that comprise a hydroxy or carboxylic acid functionality, the
releasing agent may be an ester of the analyte, which has a high
binding affinity for endogenous binding proteins relative to the
analyte to be detected and which has no significant binding
affinity for an antibody for the analyte. For example, in a
determination for sirolimus, an ester of sirolimus may be employed
as the releasing agent so long as it meets the above requirements.
It should be noted that a non-releasing agent analyte analog is a
modified analyte that does compete with the corresponding analyte
for binding to an antibody for the analyte.
[0046] A structural analog is a moiety that has the same or similar
structural or spatial characteristics as the analyte such that the
structural analog accomplishes the same or similar result as the
analog of the analyte. The structural analog may be, for example,
another compound that is related to the analyte. For example, as
mentioned above, in a determination of sirolimus, an ester of
tacrolimus may be employed as a releasing agent, or in a
determination of tacrolimus, sirolimus may be employed as a
releasing agent. The ester may be, for example, a carbamate, a
carbonate, or an ester of a C.sub.1 to C.sub.6 carboxylic acid, for
example. See, for example, U.S. Pat. No. 7,186,518, the relevant
disclosure of which is incorporated herein by reference. Other
examples of releasing agents include [Thr.sub.2, Leu.sub.5,
D-Hiv.sub.8, Leu.sub.10]-cyclosporin A for cyclosporin A, FK506 for
sirolimus, and sirolimus for FK506, for example. See, for example,
U.S. Pat. No. 6,187,547, the relevant disclosure of which is
incorporated herein by reference.
[0047] In some embodiments the releasing agent may be an agent that
disrupts cellular membranes in which the analyte is entrapped. For
example, an analyte that is entrapped within red blood cells may be
released from the red blood cells by employing a hemolytic agent. A
hemolytic agent is a compound or mixture of compounds that disrupts
the integrity of the membranes of red blood cells thereby releasing
intracellular contents of the cells and, in particular,
erythrocytes. Numerous hemolytic agents are known in the art.
Hemolytic agents include, for example, non-ionic detergents,
anionic detergents, amphoteric detergents, low ionic strength
aqueous solutions (hypotonic solutions), bacterial agents,
antibodies that cause complement dependent lysis, and the like.
Non-ionic detergents that may be employed as the hemolytic agent
include both synthetic detergents and natural detergents. Examples
of synthetic detergents include TRITON.TM. X-100, TRITON.TM. N-101,
TRITON.TM. X-114, TRITON.TM. X-405, TRITON.TM. SP-135, TWEEN.RTM.
20 (polyoxyethylene (20) sorbitan monolaurate), TWEEN.RTM.80
(polyoxyethylene (20) sorbitan monooleate), DOWFAX.RTM.,
ZONYL.RTM., pentaerythrityl palmitate, ADOGEN.RTM. 464,
ALKANOL.RTM. 6112 surfactant, allyl alcohol
1,2-butoxylate-block-ethoxylate HLB 6, BRIJ.RTM., ethylenediamine
tetrakis(ethoxylate-block-propoxylate) tetrol, IGEPAL.RTM.,
MERPOL.RTM., poly(ethylene glycol),
2-[ethyl[(heptadecafluorooctyl)sulfonyl]amino]ethyl ether,
polyethylene-block-poly(ethylene glycol), polyoxyethylene sorbitan
tetraoleate, polyoxyethylene sorbitol hexaoleate, TERGITOL.RTM.
NP-9, GAFAC.RTM. (RHODAFAC.RTM., an alkyl polyoxyethylene glycol
phosphate ester such as, for example,
alpha-dodecyl-omega-hydroxypoly(oxy-1,2-ethanediyl) phosphate), and
EP 110.RTM. and the like. Naturally-occurring detergents that may
be employed as the hemolytic agent include, for example, saponins,
sodium or potassium neutralized fatty acid, neutralized
phospholipids, diacylglycerol, neutralized phosphatidyl serine,
phosphatidate, neutralized phosphatidyl ethanolamine, phosphatidyl
choline, phosphatidyl inositol, phosphatidylcholine, bile salt,
unesterified cholesterol, neutralized sphingosine, ceramide, and
the like. Combinations of one or more synthetic detergents or one
or more naturally occurring detergents and combinations of
synthetic detergents and naturally occurring detergents may also be
employed.
[0048] Other releasing agents that may be employed in the present
embodiments include solubility reagents such as, for example, a
small amount of an organic solvent such as, e.g., methanol,
ethanol, isopropanol, methoxy propanol and DMSO; and agents for
carrying out protein digestion such as, for example, proteinases,
trypsin, pepsin, peptidases; for example.
[0049] The concentration of the releasing agent(s) in the medium is
that sufficient to achieve the desired result of displacing the
analyte from endogenous binding moieties to render the analyte and,
in some instances analyte metabolites, accessible for binding to an
antibody for the analyte as discussed above. The amount or
concentration of the releasing agent employed depends on one or
more of the nature of the sample, the nature of the analyte, the
nature of the drug metabolites, the nature of other reagent
components, and the reaction conditions, for example. In some
embodiments the amount of the releasing agent in the aqueous medium
is about 0.000001% to about 0.5%, about 0.0001% to about 0.4%,
about 0.001% to about 0.3%, about 0.01% to about 0.2%, about 0.1%
to about 0.3%, about 0.2% to about 0.5%, about 0.1% to about 0.2%,
for example (percent is weight/volume).
[0050] For some analytes such as, for example, immunosuppressant
drugs, a lysing agent such as, for example a detergent, may be
employed. The detergent makes the immunosuppressant drug more water
miscible in an aqueous assay mixture, thus rendering it more
accessible by the assay measuring agents such as assay antibodies.
In some examples, a detergent is employed that can prevent the drug
from diffusing into lipoproteins particles or other type of
liposomes. For example, PLURONIC.RTM. detergent is a detergent used
in a tacrolimus assay to prevent the drug from diffusing into the
core of lipoprotein complexes.
[0051] One or more incubation periods may be employed in the
methods in accordance with the principles described herein. An
incubation period may be applied to a medium at one or more
intervals including any intervals between additions of various
reagents mentioned above. The medium is usually incubated at a
temperature and for a time sufficient for the function that is
being carried out such as, for example, treatment with a releasing
agent, treatment with a pretreatment agent, binding of various
components of the reagents such as, for example, binding of
antibody to an analyte, to occur. Moderate temperatures are
normally employed for carrying out an incubation period. The method
may include an incubation period for one or more of the steps of
the present methods. For example, an incubation period may be
applied to one or more of the combination of the releasing agent
with the sample, the combination of the pretreatment agent with the
sample and an antibody reagent, the combination of the pretreatment
agent with the sample, an antibody reagent and a solid phase
reagent, for example. In some examples, the medium is incubated
under conditions for binding of the antibody for the analyte to
analyte suspected of being in the sample as discussed more fully
below.
[0052] The length and conditions of the incubation periods are
dependent on one or more of the nature and concentration of the
reagents, the nature of the analyte, and the suspected
concentration of the analyte, for example. In some embodiments
incubation temperatures may be about 5.degree. C. to about
99.degree. C., or about 15.degree. C. to about 70.degree. C., or
about 20.degree. C. to about 45.degree. C., for example. The time
for an incubation period depends on one or more of the temperature
of the medium and the rate of reaction or of binding of the various
reagents. The time period for the incubation is about 0.2 seconds
to about 24 hours, or about 1 second to about 6 hours, or about 2
seconds to about 1 hour, or about 1 to about 15 minutes, for
example.
[0053] Following an incubation period, if any, for binding of
antibody for the analyte to the analyte, the medium is examined for
the presence of a complex comprising the analyte and the antibody
for the analyte. The presence and/or amount of the complex indicate
the presence and/or amount of the analyte in the sample. The phrase
"complex comprising the antibody for the analyte" refers to a
complex wherein the antibody for the analyte is bound to one or
more substances that may be one or more of the analyte and other
substances in a sample that bind to the antibody for the
analyte.
[0054] In many embodiments the examination of the medium involves
detection of a signal from the medium. The presence and/or amount
of the signal are related to the presence and/or amount of the
analyte in the sample. The particular mode of detection depends on
the nature of the sps. As discussed above, there are numerous
methods by which a label of an sps can produce a signal detectable
by external means, desirably by visual examination, and include,
for example, electromagnetic radiation, electrochemistry, heat,
radioactivity detection, and chemical reagents.
[0055] The examination for presence and/or amount of the signal
also includes the detection of the signal, which is generally
merely a step in which the signal is read. The signal is normally
read using an instrument, the nature of which depends on the nature
of the signal. The instrument may be a spectrophotometer,
fluorometer, absorption spectrometer, luminometer,
chemiluminometer, actinometer, photographic instrument, and the
like. The presence and amount of signal detected is related to the
presence and amount of the analyte present in a sample if the assay
is making an accurate determination. Temperatures during
measurements generally range from about 10.degree. to about
70.degree. C. or from about 20.degree. to about 45.degree. C., or
about 20.degree. to about 25.degree. C., for example. In one
approach standard curves are formed using known concentrations of
the analytes to be screened. As discussed herein, calibrators and
other controls may also be used.
[0056] The phrase "measuring the amount of an analyte" refers to
the quantitative, semi-quantitative and qualitative determination
of the analyte. Methods that are quantitative, semiquantitative and
qualitative, as well as all other methods for determining the
analyte, are considered to be methods of measuring the amount of
the analyte. For example, a method, which merely detects the
presence or absence of the analyte in a sample suspected of
containing the analyte, is considered to be included within the
scope of the above phrase. The terms "detecting" and "determining,"
as well as other common synonyms for measuring, are contemplated
within the scope of the present invention.
General Description of Non-Agglutination Assays for an Analyte
[0057] Any suitable non-agglutination assay may be employed for
determining an analyte in accordance with the principles described
herein. The assay may be conducted on the sample as an immediate
continuation of the pretreatment as discussed above or the assay
may be carried out at a point thereafter. Thus, a particular assay
may be conducted sequentially or concomitantly. The assays are
conducted by combining the respective sample with reagents for
determining the amount of the analyte in the sample. The nature of
the reagents is dependent on the particular type of assay to be
performed. In general, the assay is a method for the determination
of the amount of an analyte in a sample. Various assay methods are
discussed below by way of illustration and not limitation.
[0058] The assay comprises adding reagents for determining the
concentration of the analyte in the sample to a medium comprising
the sample. For immunoassays, the reagents comprise at least one
antibody for the analyte or similar sbp member. An amount of a
complex comprising the antibody for the analyte is measured and the
amount of the complex is related to the concentration of the
analyte and other substances in the sample.
[0059] One general group of non-agglutination immunoassays that may
be employed includes non-agglutination immunoassays using a limited
concentration of antibody. Another group of non-agglutination
immunoassays involves the use of an excess of one or more of the
principal reagents such as, for example, an excess of an antibody
for the analyte. Another group of non-agglutination immunoassays
are separation-free homogeneous assays in which the labeled
reagents modulate the label signal upon analyte-antibody binding
reactions. Another group of non-agglutination immunoassays includes
labeled antibody reagent limited competitive assays for analyte
that avoid the use of problematic labeled haptens. In this type of
assay, a solid phase immobilized analyte is present in a constant,
limited amount. The partition of a label between the immobilized
analyte and free analyte depends on the concentration of analyte in
the sample.
[0060] Antibodies specific for an analyte for use in immunoassays
can be monoclonal or polyclonal. Such antibodies can be prepared by
techniques that are well known in the art such as immunization of a
host and collection of sera (polyclonal) or by preparing continuous
hybrid cell lines and collecting the secreted protein (monoclonal)
or by cloning and expressing nucleotide sequences or mutagenized
versions thereof coding at least for the amino acid sequences
required for specific binding of natural antibodies.
[0061] Antibodies may include a complete immunoglobulin or fragment
thereof, which immunoglobulins include the various classes and
isotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM,
etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and
the like. In addition, aggregates, polymers, and conjugates of
immunoglobulins or their fragments can be used where appropriate so
long as binding affinity for a particular molecule is
maintained.
[0062] As discussed above, an antibody selected for use in an
immunoassay for an analyte, for example, should specifically and
preferentially bind the analyte (and its pharmaceutically active
metabolites, if necessary or desired) over other ligands such as
other metabolites or related substances. Other reagents are
included in the assay medium depending on the nature of the assay
to be conducted.
[0063] As discussed above, non-agglutination immunoassays may
involve labeled reagents. Non-agglutination labeled immunoassays
include, but are not limited to, enzyme immunoassays, fluorescence
polarization immunoassays, radioimmunoassay, inhibition assay,
induced luminescence, and fluorescent oxygen channeling assay, for
example.
[0064] In some embodiments homogeneous non-agglutination
immunoassays may be employed; such assays may also be referred to
as essentially partition-free immunoassays. The present methods
have application to fully automated homogeneous assays in which,
prior to the assay, there is no extraction or separation of the
analyte from other constituents of the sample including analyte
metabolites. In a "non-manual extraction" assay, a sample such as a
whole blood sample, without extraction in, e.g., an organic
solvent, is combined with reagents for conducting an assay for the
analyte in a suitable medium and the assay method is conducted. The
present methods also find application to manual extraction
assays.
[0065] The assays can be performed either without separation
(homogeneous) or with separation (heterogeneous) of any of the
assay components or products. Homogeneous immunoassays are
exemplified by the EMIT.RTM. assay (Syva Company, San Jose, Calif.)
disclosed in Rubenstein, et al., U.S. Pat. No. 3,817,837, column 3,
line 6 to column 6, line 64; immunofluorescence methods such as
those disclosed in Ullman, et al., U.S. Pat. No. 3,996,345, column
17, line 59, to column 23, line 25; enzyme channeling immunoassays
("ECIA") such as those disclosed in Maggio, et al., U.S. Pat. No.
4,233,402, column 6, line 25 to column 9, line 63; the fluorescence
polarization immunoassay ("FPIA") as disclosed, for example, in,
among others, U.S. Pat. No. 5,354,693; and so forth.
[0066] Other enzyme immunoassays are the enzyme modulate mediated
immunoassay ("EMMIA") discussed by Ngo and Lenhoff, FEBS Lett.
(1980) 116:285-288; the substrate labeled fluorescence immunoassay
("SLFIA") disclosed by Oellerich, J. Clin. Chem. Clin. Biochem.
(1984) 22:895-904; the combined enzyme donor immunoassays ("CEDIA")
disclosed by Khanna, et al., Clin. Chem. Acta (1989) 185:231-240;
homogeneous particle labeled immunoassays such as particle enhanced
turbidimetric inhibition immunoassays ("PETINIA"), particle
enhanced turbidimetric immunoassay ("PETIA"), etc.; and the
like.
[0067] Other assays include the sol particle immunoassay ("SPIA"),
the disperse dye immunoassay ("DIA"); the metalloimmunoassay
("MIA"); the enzyme membrane immunoassays ("EMIA");
luminoimmunoassays ("LIA"); acridinium ester label immunoassays
using paramagnetic particles as a solid phase (ADVIA Centaur
immunoassays); and so forth. Other types of assays include
immunosensor assays involving the monitoring of the changes in the
optical, acoustic and electrical properties of an
antibody-immobilized surface upon the binding of an analyte. Such
assays include, for example, optical immunosensor assays, acoustic
immunosensor assays, semiconductor immunosensor assays,
electrochemical transducer immunosensor assays, potentiometric
immunosensor assays, and amperometric electrode assays, for
example.
[0068] In certain embodiments a second enzyme may be employed in
addition to the enzyme of the enzyme conjugate. The enzymes of the
pair of enzymes are related in that a product of the first enzyme
serves as a substrate for the second enzyme.
[0069] In one embodiment the assay is an induced luminescence
immunoassay, which is described in U.S. Pat. No. 5,340,716 (Ullman,
et al.), which disclosure is incorporated herein by reference. In
one approach the assay uses a particle incorporating a
photosensitizer and a label particle incorporating a
chemiluminescent compound. The label particle is conjugated to an
sbp member, for example, an antibody for the analyte that is
capable of binding to the analyte to form a complex, or to a second
sbp member to form a complex, in relation to the amount of the
analyte. If the analyte is present, the photosensitizer and the
chemiluminescent compound come into close proximity. The
photosensitizer generates singlet oxygen and activates the
chemiluminescent compound when the two labels are in close
proximity. The activated chemiluminescent compound subsequently
produces light. The amount of light produced is related to the
amount of the complex formed, which comprises antibody for the
analyte.
[0070] By way of further illustration, chemiluminescent particles
are employed, which comprise the chemiluminescent compound
associated therewith such as by incorporation therein or attachment
thereto. An sbp member that binds to the analyte, such as, for
example, an antibody for analyte, is bound to a polysaccharide
coating the particles. A second sbp member that binds to the
analyte is part of a biotin conjugate. Streptavidin is conjugated
to a second set of particles having a photosensitizer associated
therewith. The binding of the streptavidin to this second set of
particles (photosensitizer particles) may or may not involve a
polysaccharide on the particles. The chemiluminescent particles are
mixed with the respective portion of the sample suspected of
containing an analyte and with the photosensitizer particles. The
reaction medium is incubated to allow the particles to bind to the
analyte by virtue of the binding of the sbp members to the analyte.
Then, the medium is irradiated with light to excite the
photosensitizer, which is capable in its excited state of
activating oxygen to a singlet state. Because the chemiluminescent
compound of one of the sets of particles is now in close proximity
to the photosensitizer by virtue of the presence of the substances
and/or the analyte, it is activated by singlet oxygen and emits
luminescence. The medium is then examined for the amount of
luminescence or light emitted, the presence thereof being related
to the amount of the substances that bind to antibody for the
analyte or the amount of analyte.
[0071] Another particular example of an assay that may be employed
for the determination of an analyte is discussed in U.S. Pat. No.
5,616,719 (Davalian, et al.), which describes fluorescent oxygen
channeling immunoassays.
[0072] Another embodiment of an assay format is a capture assay. In
this assay format, the antibody for the analyte is covalently bound
to a magnetic particle. The sample is incubated with these
particles to allow the antibodies for the analyte to bind to the
analyte and/or substances in the sample other than analyte that
also bind to the antibody. Subsequently, an enzyme that has the
analyte or a derivative of the analyte covalently attached is
incubated with the magnetic particles. After washing, the amount of
enzyme that is bound to the magnetic particles is measured and is
inversely related to the amount of a complex comprising the
antibody for the analyte. Alternatively, the amount of enzyme in
the supernatant liquid is measured and is directly related to the
amount of a complex comprising the antibody for the analyte.
[0073] The assays discussed above are normally carried out in an
aqueous buffered medium at a moderate pH, generally that which
provides optimum assay sensitivity and does not interfere with the
assay employed. The pH for the assay medium will usually be in the
range of about 4 to about 11, or in the range of about 5 to about
10, or in the range of about 6.5 to about 9.5. The pH will usually
be a compromise between optimum binding of the binding members of
any specific binding pairs, the pH optimum for other reagents of
the assay such as members of a signal producing system, and so
forth.
[0074] Various buffers may be used to achieve the desired pH and
maintain the pH during the determination. Illustrative buffers
include borate, phosphate, carbonate, tris, barbital and the like.
The particular buffer employed is not critical, but in an
individual assay one or another buffer may be preferred. Various
ancillary materials may be employed in the above methods. For
example, in addition to buffers the medium may comprise stabilizers
for the medium and for the reagents employed. In some embodiments,
in addition to these additives, proteins may be included, such as
albumins; quaternary ammonium salts; polyanions such as dextran
sulfate; binding enhancers, or the like. All of the above materials
are present in a concentration or amount sufficient to achieve the
desired effect or function.
[0075] As mentioned above, one or more incubation periods may be
applied to the medium at one or more intervals including any
intervals between additions of various reagents mentioned above.
Conditions for an incubation period are discussed above.
[0076] The concentration of analyte that may be assayed generally
varies from about 10.sup.-5 to about 10.sup.-17 M, or from about
10.sup.-6 to about 10.sup.-14 M. Considerations, such as whether
the assay is qualitative, semi-quantitative or quantitative
(relative to the amount of analyte present in the sample), the
particular detection technique and the concentration of the analyte
normally determine the concentrations of the various reagents.
[0077] The concentrations of the various reagents in the assay
medium will generally be determined by the concentration range of
interest of the analyte, the nature of the assay, the antibody
affinity and avidity and antibody fragmentation, for example.
However, the final concentration of each of the reagents is
normally determined empirically to optimize the sensitivity of the
assay over the range. That is, a variation in concentration of
analyte that is of significance should provide an accurately
measurable signal difference. Considerations such as the nature of
a signal producing system and the nature of the analyte normally
determine the concentrations of the various reagents.
[0078] While the order of addition may be varied widely, there will
be certain preferences depending on the nature of the
non-agglutination assay. The simplest order of addition is to add
all the materials simultaneously and determine the effect that the
assay medium has on the signal as in a homogeneous assay.
Alternatively, the reagents can be combined sequentially.
Optionally, an incubation step may be involved subsequent to each
addition as discussed above.
Specific Examples of Non-Agglutination Immunoassays for
Determination of an Immunosuppressant Drug
[0079] The following specific examples are by way of illustration
and not limitation on the scope of the present invention. Selection
of tacrolimus or sirolimus as the immunosuppressant drug is also by
way of illustration and not limitation as the present invention has
general application to detection of analytes in general and
immunosuppressant drugs in particular.
[0080] The sample to be analyzed is one that is suspected of
containing one or more immunosuppressant drug analytes. In these
examples, the sample is whole blood, which is unfractionated blood
or blood that comprises both red cells and plasma. The sample is
treated with one or more releasing agents to release
immunosuppressant drug analyte from endogenous-binding substances.
For the immunosuppressant drug in this example, the releasing
agents may include one or more of a lysing agent and a hemolytic
agent as discussed above to release the immunosuppressant drug from
red blood cells. The nature and amount or concentration of
hemolytic agent and/or lysing agent employed is discussed above.
The sample and an antibody reagent are also subjected to a
pretreatment agent as described above, which may be carried out as
a separate step, or the pretreatment agent may be included in the
medium with one or more releasing agents. Thus, all of the above
may be combined simultaneously in the medium or one or more of the
above reagents may be added sequentially in concentrations as
discussed above. The medium may also comprise one or more
preservatives as are known in the art.
[0081] In one example, following treatment with one or more
releasing agents that include FK506 ester, a sample suspected of
containing tacrolimus in an assay medium is mixed with a tacrolimus
conjugate, i.e., for example, an analog of tacrolimus that is
attached to biotin. The sample is incubated to allow binding of
tacrolimus of the sample to the antibody for tacrolimus in
competition with the analog of tacrolimus where the antibody is
capable of binding to tacrolimus or the analog of tacrolimus. The
medium also includes sodium salicylate as a pretreatment agent as
discussed above. After rinsing with an appropriate wash buffer, a
detection molecule consisting of streptavidin or avidin conjugated
to an enzyme, florescent or chemiluminescent molecule or
radioactive moiety can be added to the medium, which is then
examined for the amount of signal. The amount of signal is related
to the amount of tacrolimus in the sample.
[0082] In another example, a non-agglutination immunoassay employed
is an induced luminescence assay as described above. In some
embodiments of the induced luminescence assay by way of
illustration and not limitation, the reagents include two latex
bead reagents and a biotinylated anti-tacrolimus mouse monoclonal
antibody. The first bead reagent is coated with tacrolimus or a
tacrolimus analog and contains chemiluminescent dye. The second
bead reagent is coated with streptavidin and contains a
photosensitizer dye. A medium containing a sample suspected of
containing tacrolimus and an antibody reagent is treated with
trichloroacetic acid as a pretreatment agent as discussed above,
where the medium can contain one or more releasing agents that
include FK506 to release tacrolimus from endogenous binding
substances in the sample. After conducting the assay, the resulting
chemiluminescent signal is measured at 612 nm and is an inverse
function of the concentration of tacrolimus in the sample that
binds to tacrolimus antibody.
[0083] Another specific example in accordance with the principles
described herein involves an assay format known as ACMIA (Affinity
Chromium dioxide Mediated Immuno Assay). For the ACMIA assay
format, chrome particles, which are coated with sirolimus or a
sirolimus analog, are employed as a first component. A second
component is an antibody for sirolimus. This antibody, crosslinked
to a reporter enzyme (for example, beta-galactosidase), is added to
a reaction vessel in an excess amount, i.e., an amount greater than
that required to bind all of the analyte that might be present in a
sample. Following treatment with sodium salicylate as a
pretreatment agent as discussed above and with one or more
releasing agents that include tacrolimus ester, the medium
comprising the sample suspected of containing sirolimus and the
antibody-enzyme conjugate is incubated to allow the sirolimus
analyte to bind to the antibody. Next, the chrome particle reagent
is added to bind up any excess antibody-enzyme conjugate. Then, a
magnet is applied, which pulls all of the chrome particles and
excess antibody-enzyme out of the suspension, and the supernatant
is transferred to a final reaction container. The substrate of the
reporter enzyme is added to the final reaction container, and the
enzyme activity is measured spectrophotometrically as a change in
absorbance over time. The amount of this signal is related to the
amount of sirolimus in the sample.
[0084] In a sandwich assay format, by way of example, a first
reagent comprising chrome particles coated with anti-tacrolimus
antibodies and a second reagent comprising a second antibody (or
binding protein) for the first antibody conjugated to a reporter
enzyme are employed. Following treatment with sodium salicylate as
a pretreatment agent as discussed above and with one or more
releasing agents that include FK506, a sample suspected of
containing tacrolimus and the chrome particles is incubated so that
all of the tacrolimus, if present in the sample, becomes bound to
the chrome particles. The chrome particles are washed, using a
magnet to separate the bound analyte from the supernatant. Then,
the second reagent, i.e., antibody (or binding protein) conjugated
to a reporter enzyme, is incubated with the chrome particles to
form a "sandwich". After washing, the amount of enzyme that is
bound to the chrome is measured and is related to the amount of
tacrolimus in the sample.
[0085] Another specific example of an assay format, for purposes of
illustration and not limitation, is EMIT.RTM. (Enzyme-Mediated
Immunoassay Technology). Following treatment with trichloroacetic
acid as a pretreatment agent as discussed above and with one or
more releasing agents that include tacrolimus ester, a medium
comprising the sample suspected of containing sirolimus and an
antibody for sirolimus, an enzyme conjugate, for example, a
conjugate of G-6-PDH, is incubated for an appropriate time period.
After conducting the assay, the amount of enzyme activity is
measured and is related to the amount of sirolimus in the
sample.
Kits for Conducting Non-Agglutination Immunoassays
[0086] The reagents for conducting a particular assay may be
present in a kit useful for conveniently performing an assay for
the determination of an analyte. In one embodiment a kit comprises
in packaged combination a pretreatment agent in accordance with the
principles described herein, reagents for releasing an analyte from
endogenous binding substances, an antibody for an analyte and other
reagents for performing a non-agglutination immunoassay, the nature
of which depend upon the particular assay format. The reagents may
each be in separate containers or various reagents can be combined
in one or more containers depending on the cross-reactivity and
stability of the reagents. The kit can further include other
separately packaged reagents for conducting an assay such as
additional sbp members, ancillary reagents such as an ancillary
enzyme substrate, and so forth.
[0087] The relative amounts of the various reagents in the kits can
be varied widely to provide for concentrations of the reagents that
substantially optimize the reactions that need to occur during the
present method and further to optimize substantially the
sensitivity of the assay. Under appropriate circumstances one or
more of the reagents in the kit can be provided as a dry powder,
usually lyophilized, including excipients, which on dissolution
will provide for a reagent solution having the appropriate
concentrations for performing a method or assay. The kit can
further include a written description of a method in accordance
with the present embodiments as described above.
[0088] The phrase "at least" as used herein means that the number
of specified items may be equal to or greater than the number
recited. The phrase "about" as used herein means that the number
recited may differ by plus or minus 10%; for example, "about 5"
means a range of 4.5 to 5.5.
[0089] The following examples further describe the specific
embodiments of the invention by way of illustration and not
limitation and are intended to describe and not to limit the scope
of the invention. Parts and percentages disclosed herein are by
volume unless otherwise indicated.
EXAMPLES
[0090] All chemicals may be purchased from the Sigma-Aldrich
Company (St. Louis Mo.) unless otherwise noted. Tacrolimus may be
obtained from Astellas Pharma US. Inc., Deerfield Ill.
[0091] Testing is carried out using the DIMENSION.RTM. RxL
analyzer, available from Siemens Healthcare Diagnostics Inc.,
Newark Del. The instrument is employed using ACMIA immunoassay
technology. The ACMIA assay method is described in U.S. Pat. Nos.
7,186,518, 5,147,529, 5,128,103, 5,158,871, 4,661,408, 5,151,348,
5,302,532, 5,422,284, 5,447,870, and 5,434,051, the disclosures of
which are incorporated herein in their entirety. In the embodiment
of the ACMIA method used herein and discussed in more detail below,
competition between tacrolimus analog on chrome particles and
tacrolimus in patient samples for antibody for tacrolimus
conjugated to an enzyme (the "conjugate") is utilized to determine
the amount of tacrolimus in patient samples. Conjugate that binds
to the tacrolimus analog on chrome particles is removed by magnetic
separation. The enzymatic activity from conjugate remaining in the
supernatant is measured and is directly proportional to the amount
of tacrolimus in the patient sample. In the ACMIA assay format
employed, the enzymatic activity observed when testing a sample
containing no tacrolimus is indicative of the amount of enzymatic
activity that is not bound to active antibody (i.e., cannot bind
tacrolimus on chrome particles). The enzymatic activity observed
when no chrome particle is present is indicative of the total
amount of enzymatic activity in the conjugate. These values can be
used to estimate the percent of enzymatic activity bound to active
antibody.
[0092] The following terms used herein are defined as follows:
".mu.g" is microgram(s); "mL" is milliliter(s); "mg" is
milligram(s); ".mu.L" is microliter(s); and "ng" is nanogram(s)
Example 1
Automated Non-Agglutination Immunoassay for Tacrolimus
[0093] Preparation of Pretreatment Solution.
[0094] This pretreatment solution contained sirolimus, sodium
azide, Pipes 1.5 sodium salt, EDTA disodium dehydrate, saponin,
PLURONIC.RTM. 25R2, PROCLIN.RTM. 300, neomycin sulfate and
pretreatment agent (sodium trichloroacetate, sodium salicylate or a
combination of both.
[0095] Preparation of Anti-Tacrolimus Antibody-.beta.-Galactosidase
Conjugate.
[0096] Monoclonal anti-tacrolimus antibody (clone 1H6 from Siemens
Healthcare Diagnostics Inc, Glasgow, Del.) is conjugated to
.beta.-galactosidase using a standard heterobifunctional SMCC
(succinimidyl
trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate) linker
according to known techniques. The antibody conjugate solution
contains approximately 7.5 .mu.g/mL anti-tacrolimus
antibody-.beta.-galactosidase conjugate, 30 mg/mL protease free
bovine serum albumin, 0.126 mg/mL MgCl.sub.2, 0.03 mL/mL of
ethylene glycol, 35.14 mg/mL PIPES 1.5 sodium salt, 50 mg/mL NaCl
and beta-gal mutein (inactivated beta-galactosidase), pH 6.5.
[0097] Magnetic Chrome Particle Preparation.
[0098] Tacrolimus chrome particles (immunoassay solid phase) are
prepared by conjugating tacrolimus-C22 to fluorescein, which is
used to pre-decorate anti-fluorescein antibody immobilized on
chromium dioxide particles through glutaraldehyde. The chrome
particle reagent contains approximately 2.5 mg/mL tacrolimus chrome
particle slurry, 60.8 mg/mL trehalose dihydrate and 7.2 mg/mL
CARBOWAX.RTM..
[0099] Non-Agglutination Assay for Tacrolimus.
[0100] The principle and operation of the ACMIA assay for
tacrolimus employing a pretreatment agent in accordance with the
principles described herein is as follows: 15 .mu.L of a whole
blood sample suspected of containing tacrolimus is mixed with the
hemolytic pretreatment solution in a vessel on the DIMENSION.RTM.
RxL analyzer. The whole blood is sampled from a standard cup by
first mixing the blood with the ultrasonic sample probe. The mixing
of whole blood sample with the pretreatment solution ensures the
hemolysis of the whole blood and the displacement of the
protein-bound tacrolimus molecules from their binding sites when
the sirolimus molecules were present.
[0101] Anti-tacrolimus antibody-.beta.-galactosidase conjugate (50
.mu.L) is added next to each of the reaction vessels and the
mixture is held for a period of time (10 to 15 minutes) and at a
temperature of 43.degree. C. to allow tacrolimus, if present, to
react with the antibody reagent. Chrome particles with immobilized
tacrolimus-CMO-DA10-Dexal prepared as described above are added (50
.mu.L) to each of the reaction vessels and are allowed to bind
un-bound conjugate. The tacrolimus-bound anti-tacrolimus
antibody-.beta.-galactosidase conjugate does not bind to the chrome
particles but remains in the supernatant when a magnetic field is
applied to the above reaction mixtures to separate the solution
from the chrome particles. The tacrolimus-bound conjugate is
detected by transferring the supernatant from each of the reaction
vessels to a photometric cuvette and measuring the enzymatic rate
of the conjugate in the presence of chlorophenol
red-.beta.-D-galactopyranoside (CPRG). The rate for each reaction
vessel is measured bichromatically at 577 and 700 nm.
[0102] For purposes of comparison, the pretreatment solution above
was employed without a pretreatment agent in accordance with the
principles described herein.
[0103] Sodium Trichloroacetate as Pretreatment Agent.
[0104] Table 1 shows the composition of the pretreatment solution,
which contains a pretreatment agent in accordance with the
principles described herein. Qty is quantity. EDTA is
ethylenediaminetetraacetate.
TABLE-US-00001 TABLE 1 Pretreatment Solution Name Qty (mg/mL)
Sirolimus 0.0012 Sodium Trichloroacetate 135 Sodium Azide 0.99
Pipes 1.5 Sodium salt 6.8 EDTA disodium dihydrate 0.3 Saponin 1
PLURONIC .RTM. 25R2 0.9 PROCLIN .RTM. 300 0.4 Neomycin sulfate
0.024
[0105] Table 2 shows the comparison between the pretreatment
solution containing a pretreatment agent (12% sodium
trichloroacetate) in accordance with the principles described
herein and the pretreatment solution that does not contain a
pretreatment agent. In the following tables, LC/MS.sup.2 is liquid
chromatography/mass spectrometry performed on a tandem mass
spectrometer using a reverse phase column. TCA is sodium
trichloroacetate (pretreatment agent). PT is pretreatment solution.
Control represents the use of a pretreatment reagent that does not
contain TCA.
TABLE-US-00002 TABLE 2 12% TCA Control LC/MS.sup.2 in PT No TCA in
ng/mL (ng/mL) PT (ng/mL) 11.8 11.9 10.2 5.7 5.7 4.2 2.9 2.8 2.2 0.1
-0.3 -0.1 5.9 5.9 5.1 6.8 6.7 5.1 4.8 4.8 3.9 10.5 10.8 9.8 15.1
15.6 13.9 6.8 7.4 5.9 7.6 7.7 6.3 8.2 8.2 6.9 6.6 6.7 5.6 4.1 3.8
3.1 12.3 11.7 10.6 4.1 4.1 3.3 5.9 6.0 5.3 4.8 4.8 4.3
[0106] As can be seen from the results in Table 2, the use of a
pretreatment agent in accordance with the principles described
herein obtains assay results that closely parallel those obtained
using LC/MS.sup.2 whereas the Control does not.
[0107] Sodium Salicylate as Pretreatment Agent.
[0108] Table 3 shows the composition of the pretreatment solution,
which contains a pretreatment agent (5% sodium salicylate) in
accordance with the principles described herein.
TABLE-US-00003 TABLE 3 Pretreatment Solution Name Qty (mg/mL)
Sirolimus 0.0012 Sodium Salicylate 50 Sodium Azide 0.99 Pipes 1.5
Sodium salt 6.8 EDTA disodium dihydrate 0.3 Saponin 1 PLURONIC
.RTM. 25R2 0.9 PROCLIN .RTM. 300 0.4 Neomycin sulfate 0.024
[0109] Table 4 shows the comparison between the pretreatment
solution containing a pretreatment agent (5% sodium salicylate) in
accordance with the principles described herein and the
pretreatment solution that does not contain a pretreatment
agent.
TABLE-US-00004 TABLE 4 Control 5% Salicylate in PT No Salicylate in
PT LC/MS.sup.2 (ng/ML) (ng/mL) 3.1 3.4 4.6 5.1 4.7 3.8
[0110] As can be seen from the results in Table 4, the use of a
pretreatment agent in accordance with the principles described
herein obtains assay results that closely parallel those obtained
using LC/MS whereas the Control does not.
[0111] Combination of Sodium Salicylate and Sodium Trichloroacetate
as Pretreatment Agent.
[0112] Table 5 shows the composition of the pretreatment solution,
which contains a pretreatment agent (1% sodium salicylate/11%
sodium trichloroacetate) in accordance with the principles
described herein.
TABLE-US-00005 TABLE 5 Pretreatment Solution Name Qty (mg/mL)
Sirolimus 0.0012 Sodium Salicylate 10 Sodium Trichloroacetate 110
Sodium Azide 0.99 Pipes 1.5 Sodium salt 6.8 EDTA disodium dihydrate
0.3 Saponin 1 PLURONIC .RTM. 25R2 0.9 PROCLIN .RTM. 300 0.4
Neomycin sulfate 0.024
[0113] Table 6 shows the comparison between the pretreatment
solution containing a pretreatment agent (1% sodium salicylate/11%
sodium trichloroacetate) in accordance with the principles
described herein and the pretreatment solution that does not
contain a pretreatment agent.
TABLE-US-00006 TABLE 6 1% Salicylate/ Control 11% TCA in PT No
Salicylate/TCA LC/MS.sup.2 (ng/mL) (ng/mL) 3.1 3.2 4.6 5.1 4.7
3.8
[0114] As can be seen from the results in Table 6, the use of a
pretreatment agent in accordance with the principles described
herein obtains assay results that closely parallel those obtained
using LC/MS whereas the Control does not.
[0115] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0116] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Furthermore, the foregoing description, for purposes of
explanation, used specific nomenclature to provide a thorough
understanding of the invention. However, it will be apparent to one
skilled in the art that the specific details are not required in
order to practice the invention. Thus, the foregoing descriptions
of specific embodiments of the present invention are presented for
purposes of illustration and description; they are not intended to
be exhaustive or to limit the invention to the precise forms
disclosed. Many modifications and variations are possible in view
of the above teachings. The embodiments were chosen and described
in order to explain the principles of the invention and its
practical applications and to thereby enable others skilled in the
art to utilize the invention.
* * * * *