WO1989002076A1

WO1989002076A1 - A rapid ''cowside'' immunoassay for milk progesterone

Info

Publication number: WO1989002076A1
Application number: PCT/US1988/002595
Authority: WO
Inventors: Robert S. Chiozzi; Thomas G. Adelman; Frederick J. Foley
Original assignee: Immucell Corp.
Priority date: 1987-08-27
Filing date: 1988-08-05
Publication date: 1989-03-09
Also published as: AU2312288A

Abstract

Cow milk is assayed for progesterone by first pre-incubating a first volume of milk in an otherwise empty, anti-(progesterone) antibody coated tube, and then incubating a lesser volume of that milk in that tube together with a labeled progesterone. The amount of labeled progesterone thus bound after competition with sample progesterone is compared with that amount bound in an identically handled reference tube. In the reference tube, a reference solution is used instead of the milk sample. The reference solution is not derived from milk, but has a progesterone content such that it yields the same assay result as milk with a 5-10 ng/ml progesteron content. When such a reference solution is used, consistent results are obtained even when the milk is assayed several hours after it is collected. A test tube rack means facilitates identical handling of sample and reference. Preferably, the cow milk is collected directly into the antibody-coated tube.

Description

A RAPID "COWSIDE" IMMONOASSAY FOR MILK PROGESTERONE

BACKGROUND OF THE INVENTION

Field of Invention

This invention relates to a "cow-side," rapid test for the evaluation of the estrus cycle or the indication of pregnancy in cows, which involves assaying progesterone levels in milk.

Information Disclosure Statement

Progesterone (4-pregnene-3, 20-dione) is a di-keto steroid secreted chiefly by the corpus luteum and the placenta. It is secreted into the blood, and thence finds it way into the milk. Progesterone levels are lowest at the time of heat (estrus) and highest when the corpus luteum matures, i.e., in the middle of the estrous cycle. If the cow conceives, progesterone levels remain elevated. Otherwise, progesterone levels will decline as the cycle wanes.

Some dairy farmers attempt to increase the size of their herds, and therefore their milk production, by artificial insemination of the cow as soon as possible after calving. Traditionally, dairy farmers decide which cows to inseminate by observing the members of the herd for- behavioral signs of "heat." Apparent failure of conception after artificial insemination may result from inseminating cows which were not in fact in estrus. See Foulkes, et al., Brit. Vet. J. , 138:515

(1982) . It has been reported that as many as 15-20% of cows bred were not in estrus at breeding. See Mooney, Dairy Today, 12 (Aug. 1987), Nebel, Dairy Herd Management 14 (May 1987) .

It is also desirable to be able to detect whether an insemination has been successful, i.e., whether a cow conceived and is pregnant. Typically, a veterinarian will palpate a cow 30-45 days after breeding to determine whether it is pregnant. If it is not pregnant, the dairyman must wait for the next cycle to rebreed the animal. It would be preferable to detect non-pregnancy at the 21st day mark and rebreed immediately. Rectal palpation, according to Mooney, is about 93% accurate in confirming pregnancy. However, it would be best if a preliminary screening could be used to reduce the number of examinations required. Also, the examinations are very subjective. -

It is known in the art that one may assay progesterone levels in milk to determine whether a cow is pregnant or in estrus. See Laing, U.S. 3,826,616; Baker, U»S. 4,587,212? Chang and Estergreen, Steroids, 41: 173 (1983)? Shemesh, et al., Brit. Veter. J. , 139: 41 (1983) ? Munro and Stabenfeldt, J. Endocr. 101: 41 (1984)? Foulkes, et al., Brit. Veter. J., 138: 515 (1982) . Both polyclonal and monoclonal antibodies for progesterone have been used in competitive immunoassays for this purpose.

Van de Wiel and Koops, Br. Vet. J. , 138:454 _.(1982) describe a polyclonal antibody against progesterone which is of high sensitivity (standard curve 0-20 pg? 0.5B_o of 5 pg) . However, polyclonal antibodies are less easy to manufacture in large quantities than are monoclonal antibodies, and they are less susceptible to standardization. Wright, et al.. Nature 295:215 (1982), Fantl, et al., J. Steroid Biochem. 17: 125 (1982)? White, et al., J. Clin. Endocr. Metab. 54: 205 (1982) and Booman, et al. , "Application of Monoclonal Antibodies in Animal Production: Pregnancy Diagnosis in Cattle," in HYBRIDOMA TECHNOLOGY IN AGRICULTURAL ^"AND VETERINARY RESEARCH (Sterne and Gamble, eds.: 1984).

Low progesterone levels are indicative of estrus, and high progesterone levels (in a bred animal, 19-24 days after successful breeding) are indicative of pregnancy. See Faulkes, et al. (1982) , supra? Munro and Stabenfeldt, J. Endocr., 101: 41-49 (1984)? Shemesh, et al., Br. Vet. J., 139: 41 (1983)? Chang and Estergreen, Steroids, 41: 173 (1983). However, these indications are not perfectly reliable, though progesterone tests are more reliable in detecting estrus than in detecting pregnancy.

Numerous progesterone tests are on the market or have been announced. Several of these tests (ACCUFIRM, BEST, CALFCHECK, ESTRUCHECK, ENZYGNOST, PROGESTASSAY, RPT) are reviewed by Nebel (1987). The ACCUFIRM and RPT tests represent embodiments of the present invention, and were first placed on sale in September 1987. The RPT/ACCUFIRM test are reportedly the quickest tests of those reviewed by Nebel.

The following descriptions of various commercial tests are based on the manufactures' instructions.

OVUCHECK is a "cowside" bovine milk progesterone test manufactured by Cambridge Veterinary Sciences,

Cambridge, England. It uses two controls, an estrus control and a pregnancy control. Milk is transferred into wells and "TRACER" is added. The instructions require incubation of "TRACER" for 30 minutes. Color intensity indicates the progesterone level.

PROGESTASSAY is another milk progesterone test which uses both estrus and pregnancy controls. Milk is collected in a collection vessel and a precise volume is pipetted into a test tube which already contains a progesterone-peroxidase conjugate. A monoclonal antibody-coated dipstick is placed in a test tube for 10 minutes. The dipstick is then placed into another test tube containing substrate and chromogen. The kit is made by Pitman-Moore, Inc., Washington Crossing, New Jersey.

CALFCHECK is another solid phase EIA for progesterone. The labeled species is a peroxidase- progesterone conjugate, linked together via an li- alpha-hemisuccinate group on the steroid. The chromogen is o-phenylenediamine. The solid phase is the well surface of a microtitration plate. The well surface was first coated with a donkey anti-rabbit IgG, which then binds the progesterone-specific rabbit IgG antiserum. Milk is collected in a collection bottle, rather than in the antibody-coated well. Incubation is for one hour. The standards were prepared by adding known quantities of progesterone to defatted milk. None is 5 ng/ml milk, but 2.5 ng/ml and 10 ng/ml milk standards are provided. The kit is made by Noctech, Ltd. _r Dublin, Ireland.

ESTRUCHECK is a kit manufactured by Synbiotics,

Inc. It uses an "estrus control." Milk is transferred with a dropper straw to wells coated with an anti- progesterone monoclonal antibody. A progesterone- enzyme (HRP) conjugate is added and incubated with the sample for 15 minutes. Excess conjugate is washed away and a color developing solution is added.

Aelia makes a milk progesterone assay kit including a 96-well plate coated with sheep anti- progesterone, a progesterone-alkaline phosphatase conjugate, and progesterone standards in estrus milk. Samples are pipetted into the wells. It may take "30 minutes to load a whole plate." The competitive incubation is for 15 minutes.

None of the commercial tests discussed above (other than our RPT/ACCUFIRM) come with instructions advising dairy farmers that they may or should collect the milk in the antibody-coated tubes directly or that they may or should hold the milk for as long as several hours after collection before assaying its progesterone content_* None of these tests teach pre-incubation of milk into the tube without conjugate for at least one minute. None of these teach use of incubation periods shorter than 10 minutes.

Laing, U.S. 3,826,616 describes a method of diagnosing pregnancy in milk-producing animals by assaying a progestogen.

Sadeh, U.S. 4,243,749 teaches a method of determining serum progesterone by an enzyme immunoassay.

Heap, U.S. 4,294,922 discloses a method of monitoring pregnancy in a milk-producing animal by assaying estrogen. Grochowitz, U.S. 3,841,756 describes an apparatus for milk production analysis.

Zachkheim, U.S. 3,878,831 describes a dispensing device for testing cows for mastitis.

No admission is made that any of the foregoing references constitute, prior art or pertinent prior art, that the information provided by authors of these references is correct or complete, or that the publication dates given are exact.

SUMMARY OF THE INVENTION

The present invention is directed to an improved competitive format enzyme immunoassay for milk progesterone suitable for "cowside" use.

Knowledge of the progesterone levels in the milk of a cow, relative to a reference solution, may be used for confirmation of estrus/non-estrus, detection of pregnancy/non-pregnancy, prediction of estrus, detection of embryonic or fetal death, determination of post-parturient cyclicity, identification of the luteal phase, monitoring of the progress of fertility treatment, and diagnosis of certain reproductive disorders.

It will be appreciated that it is undesirable to require the farmer to test the milk immediately after it is collected from the cows. An assay which can tolerate a delay between collection and testing is desirable since it permits the farmer greater flexibility in performing his chores. Our assay permits the farmer to run the tests four hours after collection of the milk and still obtain meaningful indications of progesterone level.

In the conventional competitive assay, both sample antigen and a known quantity of an antigen-label conjugate are incubated with an immobilized antibody.

The more antigen is present in the sample, the less of the labeled species will be bound.

In our improved assay, the milk sample is first incubated in a sample tube with the immobilized antibody without the presence of any competitive labeled species. If a large quantity of progesterone is present in the sample, many of the antigen-binding sites will be occupied. If none is present, none of the sites will be occupied (ignoring non-specific binding) .

The sample is then decanted. A minor portion of the sample will still adhere to the tube and may contain unbound progesterone. The progesterone-enzyme conjugate is added to this remnant and competes for the unoccupied antigen-binding sites with the remnant's unbound progesterone.

The use of a high-affinity antibody assures that the binding of progesterone in the initial incubation is rapid at first and gradual thereafter. Thus,, there is little difference between a ten-minute non- competitive incubation and a four-hour non-competitive inhibition.

Since a hiatus between collection and testing is acceptable, it is convenient to receive the sample into the sample tube directly from the teats of the cow. Preferably, no attempts is made to precisely quantitate the progesterone level. Rather, the sample level is compared with the level in a reference solution. This reference solution is desirably a non- milk derived solution with a progesterone level such that it behaves like a milk sample with a progesterone content exceeding that indicative of estrus and preferably in the relatively rare "mid-range" of milk samples, i.e., 5-10 ng/ml, 5 ng/ml being especially preferred. Thus, it is neither an "estrus control" nor a "pregnancy control." The actual progesterone level of the reference is actually lower (2 ng/ml) since its progesterone is more readily bound by the antibody than the progesterone in a milk sample.

To facilitate identical processing of sample and reference, the sample and reference tubes are placed in the same tube rack. Furthermore, the separate sample tubes may be filled simultaneously by a manifold means.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 compares the intensity of color development in (a) the sample tubes of a 4 tube rack holding fresh milk, (b) the sample tubes of an 8 tube rack holding fresh milk, (c) the sample tubes of a 4 tube rack holding "4-hour" milk, and (d) the sample tubes of an 8 tube rack holding "4 hour" milk, as a percentage of the color intensity developed in the reference tube of the same tube rack, with the progesterone concentration of the milk sample as determined by the validation assay (Reference Example A) . DETAILED DESCRIPTION OF THE INVENTION

In the preferred embodiment, the relative level of progesterone in milk is determined on a cowside basis using a test tube coated with a high affinity monoclonal antibody against progesterone. The milk sample is placed in this tube, and sample progesterone is bound by the insolubilized antibody. The sample is decanted, leaving a small amount of milk (about 0.115 ml) adhering to the tube walls. Immediately thereafter, enzyme-labelled antigen (Solution B) is added and competes with the sample progesterone for the antibody's binding sites. The contents of the tube are decanted to remove unreacted progesterone (sample or solution B) , the tube is rinsed, and the substrate (Solution C) and chromogen (Solution D) are added.

A non-milk reference solution (Solution A) , calibrated to behave in the assay like a milk sample with a progesterone level of about 5 ng/ml,is handled similarly.

The color development in the sample tube is then compared to that in the reference tube, preferably using a portable colorimeter.

The assay is described in more detail in Example 1, below.

Example 1: "Cowside" Competitive Immunoassay For Milk Progesterone

Preparation of Sample and Reference Tubes An antibody against progesterone, preferably one with an affinity of at least about 10¹⁰ liters/mole, is immobilized on the inner surface of a test tube. While the antibody may be coupled directly to the tube, the preferred method of immobilization is to first coat the tube with an anti-(antibody) , and then add the anti- (progesterone) antibody. Any technique of immobilizing the antibody which does not unduly impair its immunoactivity is satisfactory.

A number of monoclonal antibodies, against progesterone are known in the art. See Wright, et al. (1982)? Fantl, et al. (1982)? and White et al., (1982), supra.

Our preferred monoclonal antibody is 4 XVIII, developed by Booman, et al. (1984), supra. This is an IgM antibody raised against progesterone-11-alpha-BSA, and having an affinity constant of 2xl0¹⁰ liters/mole. Other high affinity antibodies such as Fantl's ll-P-27, may be considered. The antibody will have an affinity for progesterone which is preferably at least about 10⁹ liters/mole and more preferably at least about 10¹⁰ liters/mole. The epitope of 4XVIII is at carbon positions 17, 20 and 21 of the progesterone molecule, and is not close to positions 7 or 11. The antibody preferably should not cross-react significantly with cortisol, corticosterone, oestrone, or 17-beta oestradiol.

The "sample" and "reference" tubes are identically prepared, and these terms are used merely for convenience in distinguishing the tubes receiving sample from the tube receiving the reference solution. While it is preferable that the antibody be immobilized on the test tube, it is possible to immobilize the antibody on another support (e.g., a dipstick) and introduce that support into an uncoated test tube.

Test Tube Rack and Manifold Means

In the preferred embodiment, the test tube are placed in a test tube rack, whereby they may be simultaneously agitated in a similar manner, and simultaneously decanted (by inverting the rack) for an identical period of time.

In one embodiment, a manifold means is used to simultaneously fill the sample tubes with milk sample, and possibly also to simultaneously fill the reference solution. This manifold means may be a separate piece, or an integral part of the test tube rack.

The use of the test tube rack means will reduce differences in retained volume, while both the rack means and the manifold means will minimize variations in incubation time.

The Colorimeter

The colorimeter is preferably the colorimeter described in U.S. Ser. No. 912,953, filed September 29, 1986, incorporated by reference, herein.

Preparation of Other Reagents

Calibrator Base ■ Deionized water (60% of final batch volume) is placed in a clean, dry container. Monobasic, monohydrate sodium phosphate (NAH2PO4) (VWR Scientific) is added with stirring (9.453g/L of calibrator). Then either 8.444 g/1 (of calibrator) of sodium phosphate, dibasic, heptahydrate (Na₂HP0₄7H₂0) or 4.473 g/L (of calibrator) of anhydrous dibasic sodium phosphate, dibasic, heptahydrate (NaH₂P0₄) are added with stirring. Bovine Albumin (50 g/L batch, sigma) is added and mixed until homogeneous. When the albumin is completely dissolved, glycerol (200 ml/L, VWR Scientific is added) . Then thimerosal (200 mg/L, sigma) is added with stirring. Bring to final batch volume and adjust pH (with 6N HCl or 6N NaOH) to 6.5 + 0.5.

Solution A: Reference Solution

The calibrator base is combined with an appropriate volume of a concentrated solution of progesterone (Sigma P-O130) in ethanol to achieve a final progesterone concentration of about 2 ng/ml. As described in Example 6, such a reference solution will behave like a milk sample supplemented with about 5 ng/ml progesterone. If our non-milk reference solution in fact contained 5 ng/ml progesterone, we expect that it would behave like a milk with a 20-30 ng/ml progesterone content.

Solution B (Labeled Antigen)

Dissolve 100 mg of horseradish peroxidase (HRP) (Sigma) into 1.0 L of distilled water with gentle agitation. Add 0.75 mL of dimethylforma ide (DMF) (Aldrich) and cool to 0°C. Adjust to pH 8.0 using 2 ul aliquots of 0.01N NaOH, thus obtaining a peroxidase solution.

Dissolve 10 mg (0.023 mM) of progesterone-6- hemisucσinate (4-pregnene-6-beta-01-3 ,20-dione)

(Steraloids) in 1.0 L of DMF with gentle agitation.

Add 6.25 ul of 4-methylmorpholine (Aldrich) and cool to

-15°C. Add 6.25 ul of isobutylchloroformate (Aldrich) and agitate every 15-20 sec. for a total of 3 mins., thus, obtaining an activated progesterone.

Add the HRP solution dropwise to the activated progesterone while stirring on a magnetic stirrer. Maintain the pH between 8.0 and 10.0 by the addition of 0.05 mL aliquots of 0.1N NaOH. Cool the solution to- 15°C for 1 hour. Agitate the solution every 10-20 mins. Hold at 0°C for an additional 2 hours, continuing to agitate every 10-20 mins.

Add 10 mg of sodium bicarbonate and agitate gently to dissolve. Dialyze in 6-8K molecular size cutoff tubing for 2 days at 4°C. Change dialysate after the first 2 hours and then at least three times each day for 2 days.

Centrifuge the solution at 9000 rpm for 30 minutes. Dilute the supernatant in suitable buffer containing BSA.

Solution C (Substrate)

Place deionized water (80% of batch volume) in the container. Add sodium acetate trihydrate (13.6 g/L) with stirring. Adjust the pH to 5.5 with citric acid. Add about 0.001% thimerosal as preservative, with stirring. Add, with stirring, a 30% hydrogen peroxide solution (1.0 ml/L) . Adjust to final volume with deionized water.

Solution D (Chromogen)

To a solution of 0.1 normal hydrochloric acid containing 1 millimolar EDTA (ethylene diaminetetraacidic acid) (80% final batch volume) add Tetramethylbenzedine-HCl to a final batch volume of

0.3%. Q.S. to final volume.

Assay Procedure

Milk samples are preferably collected immediately after the cow is milked and the milking machine is removed, that is, before dipping the teats in a sanitizing solution. The residual milk is manually stripped from a clean teat directly into the antibody- coated sample tube. The dairyman may run the test immediately, or wait up until at least about four hours after the milk is collected into this tube. The sample tube which is agitated to mix milk with cream. One or more sample tubes are placed in the tube rack. Reference Solution A is placed in a tube in the reference position of the tube rack, and allowed to stand for one minute. Squeezing the ends of the rack to hold the tubes in place, the rack is inverted, thereby decanting all of the milk (and of Solution A) except for droplets adhering to the inner walls of the tube. Solution B is added to the sample tubes and the reference tube, and mixed in by shaking the rack side- to-side for ten seconds. After waiting one minute, the tubes are emptied by again turning the rack upside- down, and the tubes are washed five times with cool running water. After decanting the wash water, first Solution C and then Solution D are added to all tubes. After shaking the rack for ten seconds, a blue color develops within about 1-2 minutes.

The ^result may be read by eye, or with a reader device. When reading by eye, when a sample tube develops a darker blue than the solution in the reference tube, a low progesterone level (associated with estrus) is indicated. When it is lighter, a high progesterone level (associated with non-estrus or pregnancy) is demonstrated. The test for pregnancy is performed 19-24 days after insemination. Pregnancy should be confirmed by veterinary examinations.

Reference Example A: Validation Assay

A mouse monoclonal antibody against progesterone (antibody 4XVIII) is bound to standard microtiter wells of a microtiter plate (Nunc-Immuno Plate II) via an intermediate sheep anti-(mouse Ig) antibody (SAM) . The SAM is incubated with shaking for at least 15 hours? the anti-progesterone antibody is then incubated with shaking for two hours. The plates are then coated with .8% BSA in assay buffer for at least 45 minutes, inverted, and air-dried.

A set of standards (10 mg/ml, 1 mg/ml, 200 ug/ml, 8 ug/ml, 800 ng/ml, 80 ng/ml, 8 ng/ml, 1 ng/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml) is prepared by making dilutions of progesterone (Sigma P-0130) in ethanol and 2% progesterone-free milk in -assay buffer (0.8 ml thoroughly mixed milk from day 0 estrus cows, 39.2 ml assay buffer) . Unknowns are added in duplicate to the microtiter wells (100 ul/well) after dilution to 2% (1:50 dilution) in assay buffer. Standards are added in quadruplicate (100 ul/well) .

Conjugate (25 ul) at an appropriate dilution is added to each well immediately after the well is loaded with unknown or standard. The plate is incubated with shaking for 60-90 minutes at room temperature. Plates are washed five times with 0.05% TWEEN 80 and substrate solution (150 ul/wells, 30-60 minutes incubation at room temperature) is added. The reaction is halted with stop solution (50 ul/well) and the optical densities are measured at 450 nm in a microtiter spectrophotometer. Progesterone concentrations in unknown are computed from the standard curve (optical density vs. ng progesterone/ml milk) .

Assay buffer is 1.265g NaH₂P0₄.H₂0, 9.06g NaH₂P0₄.7H₂0, 9.5g HCl, 0.2g Thimerosal (Sigma T-5125) and l.Og Albumin, fraction V (BSA) (Sigma A-4502) in 1000 ml deionized water. Substrate solution is 24.5 ml of 0.1M acetate-citrate (pH 5.5), 100 ul 1% H₂0₂, and 400 ul of TMB stock solution (10 ml dimethyl sulfoxide (DMSO) , Sigma D-5879, and 60 mg tetra-methylbenzidine, TMB, Sigma T-2885) . Stop solution is 11 ml H₂S0₄ in 89 ml deionized water. Conjugate is horseradish peroxidase conjugated to progesterone at carbon atom 6.

Example 2: Assay Accuracy In Determining Sample

Progesterone Level Relative To The Reference Level

Milk samples were taken at several farms. Each sample was divided in half, and one half was tested by RIA, the other by the instant assay. The RIAs were performed by academic institutions and the EIAs described herein by dairymen.

TABLE 1: FIELD ACCURACY OF ELISA MILK PROGESTERONE (RPT)

No. Samples in Percent

Farm sagreement Accurac

BJB

JF

JTH

LLM

RHF

ES

CMS

DS

RDW

WW

TOTAL

The 9.7% divergence between the present assay and the RIA may be in part attributable to errors in the validation assay, sample collection errors, mislabeling, and the use of both day 0 and day 21 (postbreeding) samples, etc.

Example 3: Distribution of Progesterone Levels in Milk Samples

A total of 1593 milk samples, all post-milking strippings collected from local farms without regard to whether the animal was "open" or was pregnant, were tested by the validation, assay (Ref. Ex. A) for progesterone content, with the following results:

Low Range 36%

(0-4 ng/ml)

Mid-Range 6%

(5-10 ng/ml)

High Range 58% (11+ ng/ml)

This shows that few milk samples had progesterone levels in the range of 5-10 ng/ml.

A more detailed analysis of the progesterone content of the milk samples appears in Table 2 below:

Example 4: Kinetics of Binding of Progesterone to High-Affinity Monoclonal Antibody

Milk was introduced into two tubes ("sample tubes") , held in a four tube rack for 5 seconds to 4 hours, and then decanted. A reference solution was added to a third tube ("reference tube") one minute before the milk was decanted from the sample tube. A final tube ("blank tube") was left empty. All of the tubes were decanted by inverting the tube rack and each tube was rinsed thoroughly with deionized water (5 times) . Thus, there was no residual milk left in the tubes which might contain unbound progesterone. Conjugate was added to each tube (including the blank) for 1 minute. (Because of the prior washing step, the conjugate was not competing with free milk sample progesterone for antibody binding sites.) The tubes were then washed five times with deionized water. Substrate and chromogen were added and the color generating reaction was allowed to continue for 2 minutes before being stopped with sulfuric acid. Tubes were read in a spectrophotoraeter, and the data was normalized by assigning the value of 100% to the signal read in the blank tube (conjugate only, one minute) .

When milk was pre-incubated with the immobilized antibody prior to addition of conjugate, the amount of conjugate bound in the subsequent one minute incubation was 65-70% of that bound when conjugate was added for one minute to a tube not pre-incubated with milk.

Surprisingly, a four hour pre-incubation only reduced this level to about 60% of the blank. This shows that most of the binding of milk progesterone to antibody

(absent a competitive species) occurs within the first minute of incubation, and that leaving the milk .sample in the sample tube for longer periods such as four hours will not greatly change the number of occupied progesterone binding sites.

Example 5: Effect of Incubation of Milk in Antibody- coated Tube for Various Time Periods on Apparent Progesterone Levels in Competitive Immunoassay Progesterone levels of sets of four and eight tubes, all save one tube in each set containing milk samples (the last containing reference solution) were assayed as described in Example 1. The milk samples were either assayed when fresh, or after being held in the antibody-coated sample tubes for four hours.

Figure 1 relates the optical density of the milk samples (after-competitive binding and color- generation) as a percentage of the optical density of the identically treated reference solution (ordinate) to the progesterone concentration (ng/ml) of the milk sample as determined by the validation assay (Reference Example A) .

It will be seen that in the four tube assay, when the progesterone level was less than or equal to about 4 ng/ml, the color in the sample tubes was more intense than that in the reference tube, regardless of whether the milk had been fresh or pre-incubated in the sample tube for four hours. Moreover, when the progesterone level was greater than about 9.5 ng/ml, the color in the sample tube was less intense than that in the reference tube, with the same disregard for the period of pre-incubation. Similar results, were obtained with the 8 tube assay, but the critical values were about 4.5 ng/ml and about 12.5 ng/ml. The greater spread is probably attributable to the fact the first tubes filled had more opportunity to bind progesterone than the last tube filled, and the period needed to fill all the tubes was greater for the 8 tube assay.

Since 94% of the milk samples reviewed in the experiment reported in Example 2 were outside the range of 5-10 ng/ml, where the period of preincubation affected whether the sample signal was greater or less than the reference signal, it follows that the reference solution employed makes it possible to provide an assay for milk progesterone which may be applied to samples collected hours before and still provide useful results.

Few samples, as noted, are in the range of 5-10 ng/ml. Indeed, most are less than 3 ng/ml or in the range of 15-20 ng/ml. When "low" and "high" progesterone samples are compared for both fresh and "4 hour" milk samples, we find that the ratio of signal levels (color intensities) of low-to-high samples are at least as good for 4 hour milk as for fresh milk.

Indeed, when samples with less than 3 ng/ml are compared with those in the range of 15-20 ng/ml, discrimination is actually improved by assaying the milk four hours after collection in the antibody-coated sample tube.

Example 6: Development of Reference Solution

A non-milk progesterone solution is a better reference solution because it is well defined. However, in a competitive immunoassay, a milk standard and a non-milk reference solution of identical progesterone levels will not behave identically. This is attributable to the fact that milk contains a variety of proteins and other substances which may bind to the antibody or to the progesterone and thereby interfere with the course of the assay.

Progesterone was added to day 0 estrus milk to provide a milk standard with a 5 -ng/ml level. This standard was compared with a series of non-milk reference solutions with different levels of progesterone. The 2 ng/ml non-milk reference assayed similarly to the 5 ng/ml milk standard. This is the most preferred reference level for the reasons set forth in Example 5. However, reference solutions behaving like milk standards with 5-10 ng/ml progesterone, are more generally preferred since few milk samples fall in that range.

While an enzyme assay is preferred, other signal- generating systems, known in the assay art may be employed. Thus, the progesterone may be tagged with a radioisotope, or attached to a fluorophore, a latex particle, colloidal gold, an iron chelate, or a magnetic particle. The color-generating reaction may be the result of the action of a single enzyme on a substrate, of the reaction of a first enzymatic product with a chromogen, or of the action of an enzyme on a substrate where the substrate is itself the product of an earlier enzymatic reaction.

The label may be conjugated directly to the progesterone, or by means of a biotin-avidin, biotin- streptavidin, peroxidase-anti-(peroxidase) , lectin- carbohydrate, liposome, or other coupling moiety.

While it is preferable to merely decant most of the "pre-incubation" milk from the tube and then incubate the conjugate with the remaining droplets, it is also possible to wash out the tube entirely and then add a few drops of milk. Yet another possibility is to supply the antibody on a dipstick with a fiber matrix, and transfer the dipstick to a waiting tube of conjugate. The matrix will retain a small volume of milk.

Claims

1. A method of determining whether a milk progesterone sample has progesterone level less than or greater than the apparent level of a reference solution which comprises:

(a) providing a monoclonal antibody against progesterone, said antibody being immobilized on the inner surface of a sample tube and on the inner surface of a reference tube?

(b) incubating a milk sample in said sample tube and a reference solution in said reference tube whereby, if the sample contains progesterone, at least some of said progesterone becomes bound and whereby at least some of the progesterone-binding sites of said antibody are thereby occupied?

(c) decanting all save a minor retained portion of said sample from said sample tube, and of said reference solution from said reference tube?

(d) introducing a labeled progesterone conjugate into the sample tube, and into the reference tube, whereby the conjugate and previously unbounded progesterone in the retained portion compete for the remaining progesterone binding sites of said antibody? and

(e) comparing the amount of labeled progesterone bound to the sample tube with the amount of labeled progesterone bound to the reference tube.

2. The method of claim 1 wherein the incubation of step (b) is performed for at least about five minutes.

3. The method of claim 1 wherein the antibody has an affinity for progesterone of at least about 10⁹ liters/mole.

4. The method of claim 1 wherein the antibody has an affinity for progesterone of at least about 10¹⁰ liters/mole.

5. The method of claim 1, wherein the reference solution is not derived from milk.

6. The method of claim 1, wherein the reference solution has an apparent progesterone level exceeding that indicative of estrus.

7. The method of claim 5, wherein the reference solution has a progesterone content such that it behaves immunologically like a milk sample with a progesterone content in the range of about 5 to about 10 ng/ml.

8. The method of claim 1, wherein said tubes are placed in tube rack means whereby they may be simultaneously agitated and simultaneously decanted.

9. The method of claim 1, wherein the milk sample is expressed from one or more of the teats of the cow directly into said sample tube.

10. The method of claim 1 wherein the label is an enzyme, and where, in the comparison step (e) , a substrate for the enzyme is provided.

11. The method of claim 1, wherein said tubes are simultaneously agitated and simultaneously decanted by placing the tubes in a tube rack means and shaking or inverting the tube rack means.

12. The method of claim 11 wherein the sample tubes are simultaneously filled with milk sample or with conjugate through manifold means having a plurality of outlets, each outlet communicating with an inlet of a sample tube.

13. A method of evaluating whether a cow is in estrus which comprises determining the level of progesterone in the milk of said cow by the method of claim 6.

14. A method of evaluating whether a bred cow is pregnant which comprises determining the level of progesterone in the milk of said cow. by the method of claim 1, wherein the reference solution has an apparent progesterone level exceeding that indicative of estrus and beneath that indicative of pregnancy.

15. The method of claim 1, wherein the competition step (d) is performed for no more than about five minutes.