Direct enzyme immunoassay for measurement of serum progesterone levels

Patent metadata

Field	Value
Publication number	US-9201077-B2
Application number	US-84423110-A
Country	US
Kind code	B2
Filing date	Jul 27, 2010
Priority date	Jul 27, 2009
Publication date	Dec 1, 2015
Grant date	Dec 1, 2015

Abstract

Compositions, methods and kits for determining progesterone levels in mares are disclosed.

First claim

What is claimed is:

1. A direct competitive enzyme linked immunoabsorbent assay (ELISA) for determining progesterone concentration in a sample taken from an equine test subject, comprising:
a) providing in at least one container immobilized antibody that specifically binds to and is specific for progesterone;
b) obtaining at least one plasma or serum sample from said test subject,
c) treating each of said plasma or serum sample(s) by (i) mixing a volume of the sample with a volume of acid which reduces the pH of the sample to about 4 thereby releasing progesterone from cortisol binding globulin and albumin present in said sample, and (ii) adding to the acidified sample a volume of assay buffer, the combined volumes of said buffer and said acid being equal to the volume of said sample before acidification, thereby providing a 1:1 dilution of the treated sample;
d) contacting the immobilized antibody in one of said container(s) with an aliquot of one of the treated sample(s) resulting from step c), said aliquot having a volume sufficiently low to provide accuracy and precision of the assay;
e) introducing into the sample-contacted container(s) a known amount of tracer reagent, in solution, comprising progesterone operably linked to an enzyme, the progesterone moiety of the tracer reagent capable of being specifically bound by the immobilized antibody and said linked enzyme moiety capable of producing a detectable signal indicative of progesterone concentration when reacted with a substrate therefor;
f) incubating the contents of the sample container(s) under conditions promoting competitive binding of progesterone in the tracer reagent and treated sample(s) to said immobilized antibody;
g) removing unbound tracer reagent, if any, from the contents of the sample container(s) after step f) via washing;
h) adding said substrate to the washed container(s) of step g);
i) measuring the detectable signal(s) produced in the container(s) of step h) from the enzyme-substrate reaction(s); and
j) correlating the measured detectable signal(s) with at least one standard containing a known concentration of progesterone, thereby determining the progesterone concentration in each of said sample(s).

2. The assay of claim 1 , wherein the immobilized antibody is an affinity-purified rabbit anti-progesterone antibody raised against progesterone 11α-hemisuccinate-bovine serum albumin (BSA).

3. The assay of claim 1 , wherein the tracer reagent comprises horseradish peroxidase (HRP) conjugated to a 3-O-carboxymethyloxime (3-CMO) derivative of progesterone.

4. The assay of claim 1 , wherein the substrate is a tetramethylbenzidine (TMB) substrate and said enzyme is streptavidin horse radish peroxidase.

5. The assay of claim 1 , wherein said acid is 1N HCl.

6. The assay of claim 1 , wherein the container is a cuvette in which said antibody is immobilized.

7. The assay of claim 6 , wherein said assay determines progesterone concentration in a range of 0 to 20 ng/ml.

8. The assay of claim 6 , wherein the detectable signal(s) are measured by placing said cuvette(s) in a spectrophotometer.

9. The assay of claim 1 , wherein the antibody is immobilized in wells of a microtitre plate.

10. The assay of claim 1 , wherein step d) and step e) are carried out simultaneously.

11. The assay of claim 1 , wherein step d) and step e) are carried out sequentially.

12. The assay of claim 1 , wherein the aliquot volume in step d) is 0.5% of the volume of the treated sample resulting from step c).

13. The assay of claim 1 , wherein correlating the detectable signal(s) with the concentration of progesterone in at least one standard is by reference to a standard curve.

14. The assay of claim 13 , wherein the standard curve is derived from a series of standard solutions determined in the manner of steps c-i), with each standard solution containing a different predetermined concentration of progesterone within a range of 0-20 ng/ml.

15. The assay of claim 1 , wherein the assay determines progesterone concentration in a range of 0 to 4 ng/ml.

16. The assay of claim 1 , wherein the concentration of progesterone determined in each of said sample(s) is compared to a predetermined progesterone concentration level associated with a parameter selected from the group consisting of normal equine luteal values, confirmation of ovulation, presence or absence of anovulatory follicles, identification of transitional mares, evaluation of progesterone augmentation therapies, evaluation of embryo transfer recipients, evaluation of progesterone levels in pregnant mares, activity of secondary corpora lutea , and failure of maternal recognition of pregnancy.

Assignees

• Nett Terry M
• Mccue Patrick M
• Brooks Ryan M
• Bruemmer Jason E
• Denniston David J
• Univ Colorado State Res Found

Inventors

• Nett Terry M
• Mccue Patrick M
• Brooks Ryan M
• Bruemmer Jason E
• Denniston David J

Classifications

• G01N33/551

  the carrier being inorganic · CPC title

• C07K16/26

  against hormones {; against hormone releasing or inhibiting factors} · CPC title

• G01N33/5306

  Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding · CPC title

• G01N2333/575

  Hormones · CPC title

• G01N33/543

  with an insoluble carrier for immobilising immunochemicals · CPC title