MXPA00007527A - Method and apparatus for detecting conception in animals - Google Patents

Abstract

The present invention provides antibodies which specifically bind early conception factor, which can be found in body fluids of animals including but not limited to the cow, cat, dog, horse, human, sheep, and pig. The invention provides methods for detecting conception or the absence of conception in an animal, the latter being recognized by the absence of early conception factor in a suitable body fluid collected from the animal. Apparati for detecting early conception factor in a body fluid from an animal comprising the antibodies which specifically bind early conception factor are also provided.

Description

METHODS AND APPARATUS FOR THE DETECTION OF CONCEPTION IN ANIMALS FIELD OF THE INVENTION This invention relates to the field of detection of conception and / or implantation in animals, including humans and devices for the same.

BACKGROUND OF THE INVENTION It has been a widely sought objective of physicians and veterinarians to have reliable markers for the diagnosis of conception, implantation and viable pregnancies, to help the management of the treatment of infertility and the treatment of pregnancy in its initial stage. . In humans, pregnancy in its initial stage diagnosed based on placental protein markers is only routinely reliable in the 4 weeks after conception and the ultrasonic analysis is only reliably positive at 7-8 weeks of gestation.

In the livestock industry, it is important to be able to identify animals that have not been successfully conceived following fertilization. For example, in the cattle industry currently, there is no route to identify such animals before 35-40 days after fertilization and the identification should be done by a veterinarian using touch. Alternatively, ultrasound analysis can detect embryonic development in 21 days. Veterinary touch is by far the most commonly used method, costing approximately $ 4 to $ 10 per test. The cost of ultrasound analysis is prohibitive for the routine management of a farm. In addition to the cost of these tests, the farmer suffers a significant financial loss additional to having cattle that have been raised but have not been conceived, also referred to as "open" cattle. For the farmer the "open" cattle have an additional cost of 4 to 10 dollars per day. The best milk producers are the most difficult breeding animals, even if they are "open", the loss is even greater. Less than 50% of the cattle conceive in the first fertilization. Due to this factor, the usual fertilization program allows 2 A semen loads per animal. If the time interval during which the animal is "open" can be shortened to days instead of months, this would substantially increase the total ranching range of the cattle.

A factor, called the pregnancy factor in its initial stage (EPF) or more recently the pregnancy factor in its initial immunosuppressive stage (ISEPF), have been detected in animals that use a bio-test and are thought to be responsible for the suppression of the maternal immune response against the embryo / fetus. Despite the demonstration of the activity through the bio-test, the current literature differs from the MW forms for the ISEPF. In mice, Clarke et al. (Clin Exp. Immunol., 32: 318, 1978) reported an EPF of approximately 180,000 kD. In sheep, Clarke et al (J. Reprod Immunol 1980 Vol.2: 151) described the existence of multiple forms of EPF, including 20 kD, 50 kD and 250-350 kD forms. In 1987, in a paper for the same laboratory, Clarke et al described the purification of a 12 kD EPF from the placenta of a sheep at 12 weeks of pregnancy (J. Reprod, Immunol, 1987, 10: 133-156). Cavanaugh describes the purification of a 21 kD EPF from the cultured ovaries and oviducts of mice, which is composed of three subunits, size 10.5, 7.2 and 3.4 kD (J. Reprod. Fertil 71: 581, 1984). The factor has been described more recently as a high molecular weight glycoprotein (Therlfall, 1993), but prior to this invention, a functionally pure preparation was not known.

An indirect bioassay for ISEPF uses an in vitro rosette inhibition test described by Bach and Antoine (Nature 217: 658 1968), which measures the ability of ISEPF to improve the inhibition of rosette formation between T cells and induced heterologous erythrocytes using anti-lymphocyte serum (ALS). Both molecular weight components must be present to detect the ISEPF in the rosette test. It has been postulated that ALS sterically hinders the binding of erythrocytes in the test: ISEPF improves inhibition by saturation of some binding sites in lymphocytes (Smart, YC et al., Fertile &; Steril 35: 397, 1981): ISEPF has been found in the mouse (Morton et al., Nature 249: 459 1974), rats (Heywood, LH et al., Australian Soc. For Reprod. Biol. 1979), humans ( Morton, H. et al., Lancet 394 1977), sheep (Morton, H. et al. Res. In Vet. Sci. 26: 261 1979), pigs (Grewal, AS et al., Australian Soc. For Reprod. Biol. 1981), and cattle (Nancarrow et al., J. Reprod. &Fert. 57: 385 1979). Noonan et al (Nature 278: 629 and 649 1979) has described ISEPF as non-specific species.

Given the appearance of ISEPF very soon after mating, it is possible that ISEPF may be an excellent initial marker for conception in animals. However, the Rosette inhibition test is technically difficult to perform, due to time consuming, uncomfortable and subject to numerous false positive readings (Sinosich et al., 1985). To develop an ISEPF test that is reproducible and is not subject to a greater number of false positive signals, a substantially pure preparation of ISEPF is required. Prior to this invention, protocols for the complete purification of a high molecular weight ISEPF had not been reported.

There is still a need for a reliable test to detect pregnancy as soon as possible after conception and also to detect miscarriage.

There is a need to raise animals and determine in 12-48 hours, if fertilization has resulted in conception. In cattle, as an example, such animals that do not conceive can be recycled with prostaglandin injections and inseminated again without the loss of thirty days. There is more in depth a need to be able to improve the capacity of the selected cattle to implant in a higher range.

SUMMARY OF THE INVENTION The present invention provides a purified factor, herein referred to as the "initial conception factor" or ECF, antibodies specific for the ECF and the equipment and apparatus for detecting the presence or absence of ECF in fluids or samples of tissue taken from animals. Methods for the detection of the conception in the 12-48 hours of fertilization / mating are described. Methods for the detection of fetal death followed by conception and implantation are also provided. Means are also provided for the improvement of embryonic implantation using the ISEPF and the anti-ISEPF antibodies of this invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic of the support of the present invention, showing 1) a support in which a sample containing ECF has been analyzed ("ECF-positive") and 2) a support in which a sample that does not contain ECF has been analyzed ("ECF-negative"); "T" marks the location of the test band or the anti-ECF antibodies; "C" marks the location of a band of control antibodies. Figure 2 is a diagram of a body in contact with a support of the present invention, shown in open and closed positions.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used in this document, "conception" can be used interchangeably with "fertilization". As used in this document, "one" can mean one or more than one.

As used herein, "purified" refers to a protein (polypeptide, peptide, etc.) that is substantially free of contaminants or cellular components with which it normally occurs to distinguish it from contaminants or other components of its natural environment. The purified protein may be homogeneous, but it needs to be non-homogeneous. It must be sufficiently free of contaminants to be useful in a clinical investigation, for example, in a test to detect protein antibodies.

Detailed Description The present invention provides antibodies that specifically bind to purified ECF. The antibodies can be reactive specifically with a single epitope of the antigen or can also react with epitopes from other organisms. The term "link" means capable of reacting or otherwise associating non-randomly with an antigen. "Specifically binding", "specifically reactive with" or "specifically against" describes an antibody or other ligand that does not cross-react substantially with any antigen other than a specific one, in this case, the purified ECF. Preferably, the purified ECF has a molecular weight between 190,000 and 205,000 thus measured by denaturing gel electrophoresis in a gradient of 4-15% polyacrylamide gel, with appropriate MW standards including 20,000, 144,000 and 208,000 [Amersham Poliacril standars®]. The ECF glycoprotein is obtained through an initial purification step that removes all non-glycoproteins. This step can be extraction with perchloric acid. The resulting glycoprotein fraction can be used as described herein to produce antibodies and to treat animals, including humans. The ECF can further be purified by ion exchange chromatography and again again by column chromatography, resulting in Fractions A1, A2 and B, as described in Example 1. These reactions are combined to produce a further purified ECF. The ECF purified by one or more steps described in Example 1 and Fractions A1, A2 and B thereof, can be obtained from cattle, cats, dogs, humans, horses, sheep and pigs.

The present invention provides antibodies that can recognize and bind ECF from cattle, cats, dogs, humans, horses, sheep and pigs. The antibodies present can be of any isotype, for example, IgG, IgM or IgA types, of any animal and can be polyclonal, monoclonal, humanized, fully human or chimeric. The antibodies can be divalent or monovalent single chain antibodies. As contemplated herein, the antibody includes any ligand that binds to the ECF, for example, an intact antibody, a fragment of an antibody or other reagent that has reactivity with the antigen. Antibodies raised against the ECF of one species can be used to recognize and bind the ECF of another species. The optimization of antigen-antibody interspecies reactions is carried out in accordance with the protocols known in the art, including the optimization of the antigen-antibody ratio and the blocking of proteins used to improve the specificity. Preferably, antibodies raised to the ECF of a given species are used to recognize and bind the ECF of the same species.

The present invention provides a method for the detection of the ECF glycoprotein using antibodies, by contacting a fluid or tissue sample of the subject with an amount of anti-ECF antibody specifically reactive with ECF and detecting the reaction. It is contemplated that the ECF can be detected in an intact form in the sample or as fragments. The sample fluid of this method can comprise any bodily fluid containing ECF or an ECF containing cell, such as blood, plasma, serum, saliva and urine. Other possible examples of body fluids include sputum, mucus, gastric juices and the like. An effective method for the detection of the ECF can, for example, be as follows: (1) bind the anti-ECF antibody to a support, (2) contact the antibody binding to a sample fluid or fluid containing ECF, (3) ) contact the above with a secondary antibody bound to a detectable part (eg, equine peroxidase enzyme or alkaline phosphatase enzyme), (4) contact the above with the substrate for the enzyme, (5) contact the above with a reagent from color, (6) observe the color change. In a specific embodiment, the washing steps include one or more stages listed above. The detectable part will allow visual detection of a precipitate or a color change, visual detection by microscopy or automatic detection by spectrometry, radiometric measurement or the like. Examples of detectable parts include fluorescein and rhodamine (for fluorescence microscopy), equine peroxidase (for electron or light microscopy and biochemical detection), biotin-streptavidin (for electron or light microscopy), colloidal gold (for formation of precipitates ) and alkaline phosphatase (for biochemical detection by color change). The methods of detection and the parts used can be selected, for example, from the above list or other appropriate examples by the standard criteria applied to said selections (James W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1983 and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988).

A specific embodiment of this invention for detecting ECF involves the use of a "penetration band" test in which the band (the "penetration band") is prepared with anti-ECF antibodies immobilized in the band in a cut band. in a location spatially separated from the location for sample loading. At the location of the sample charge, labeled anti-ECF antibodies are deposited with a detectable part. Subsequently, a) a test fluid is added to the sample loading pad, b) the ECF in the test fluid binds to the anti-ECF antibodies and this complex passes along the band by capillary flow until contacts the band of the immobilized anti-ISEPF antibody and c) the ECF anti-ECF complexes are concentrated in the band, allowing the visualization of the detectable part by any of the methods described above. In a further embodiment of this invention, a second band of antibodies can be immobilized in the band, on the distal side of the sample loading band. The antibodies of this second band are selected to recognize the immunoglobulin of the animal in which the anti-ECF antibodies were produced, for example, goat anti-IgG. This second band serves as an internal positive control for the penetration band test to demonstrate that the penetration band works properly. The amount of anti-ISEPF antibodies immobilized in the ppmera band are controlled so that sufficient ECF complex: anti-ECF antibody will move through the location of the first band to contact goat anti-IgG antibodies.

The present invention provides a method of detecting the conception or implantation in an animal that comprises detecting the presence of ECF in samples taken from a paired female animal. The type of sample taken will depend on the species of animal being tested, but serum, urine or milk samples are preferred. You can also use vaginal secretions and saliva. The sample can be used without further process can be processed by dilution in a fluid, such as a blocking solution for non-specific bound binding. Examples of blocking solutions include SeaBlock (East Coast Biologicals, New Brerwich, ME) mixed with 1% newborn calf serum and 10mM Tris with 2% Tween-20.

The present invention further provides a method for detecting the absence of conception in an animal in the 12-48 hours of pairing comprising the determination of the presence or absence of ECF, the absence of ECF indicating the absence of conception.

The present invention provides methods for the detection of spontaneous abortions in pregnant animals by monitoring the level of ECF after mating, for example, from less than one day to 48 days. Preferably, the ECF levels are monitored periodically following the conception and / or implementation. In humans, such monitoring can also be used to minimize the use of abortion-inducing drugs, for example, RU-486, by indicating whether conception and implantation has followed mating.

The present invention can be used to improve the likelihood of implantation or conception in animals, including humans and to minimize the chances of abortion. Low levels of ECF correlate with a low probability of conception or implantation, while higher levels of ECF are a good predictor of a high probability of conception, implantation and maintenance of pregnancy. Therefore, animals could be provided with additional ECF, most likely through intravenous administration, to place their ECF levels at the appropriate ranges for conception, implantation and maintenance of pregnancy.

The present invention provides an apparatus for use in detecting the presence of ECF comprising a support in which the ECF antibodies are present. In one embodiment (Figure 1), the support (1) comprises a band made of matepal along which the flow can flow. At one end of the band, the sample fluid is introduced and the fluid flows along the support and contacts the antibodies to the ECF. In a specific embodiment, a filtration aid (2) such as Whatman CHO-17, is attached to a non-sampling end of the band to improve fluid flow. In a specific embodiment, the location for the introduction of the sample includes an absorbent pad (3) which may be made of a glass fiber material, Whatman FO-75 or any other suitable material.

The present invention provides an apparatus for use in the detection of the presence of ECF comprising a body in contact with a support in which the ECF antibodies are present. The body can be of different types of materials, for example, plastic, metal or cardboard and can be in any way that will accommodate the support. A specific embodiment (Figure 2) of the body (4) is a compact balloon-shaped 4 to 10 cm long, hinged at one end with guides (5) to secure the support and an opening (6) for the introduction of the sample and another opening (7) to see the antibody-antigen reaction. Another specific modality is a box in rectangular shape with upper and lower end (2-3) x (4-10) cm. The support can be of any material to which antibodies can bind. Different types of antibodies can bind better to one support than to another, as is well understood in the art. As an example, IgA monoclonal antibodies do not bind well to most membranes. However, having determined a method for accomplishing this, the present invention can use IgA antibodies.

In one embodiment of the apparatus, the ECF antibodies are monoclonal IgAs and the sample pad is a glass fiber matepal. In a specific embodiment, the absorbent sample pad is made of Whatman FO-75. In a specific embodiment of the apparatus the ECF antibodies are polyclonal and the support is a 5 micron nitrocellulose membrane. In a specific embodiment, the nitrocellulose membrane is Whatman 5 μM. Other membranes, currently available or developed later, can also be used. The absorbent sample pad and any filter aid can be disposed on the upper part of the support material, for example the nitrocellulose membrane. Alternatively, the absorbent sample pad and a filtration aid may be positioned so that their ends border the ends of the support, such as the nitrocellulose membrane.

In one embodiment of the apparatus, the support will have anti-ECF antibodies placed in two spatially separated locations. A fluid contains the test sample of an animal that is introduced to the support and the support filters the fluid so that it first contacts the location on the support where the labeled antibodies have been deposited and subsequently continues filtering through the support to contact the second location where the antibodies are linked. Anti-ECF polyclonal antibodies can be used in both locations or any combination of monoclonal and polyclonal anti-ECF antibodies can be used in two locations. For example, anti-ECF labeled monoclonal antibodies are placed in a location on the support, polyclonal anti-ECF antibodies are bound to the other location and the test sample is introduced at the location where the monoclonal antibodies have been placed. In another embodiment, the anti-ECF labeled polyclonal antibodies are placed at the location where the sample was introduced and the anti-ECF monoclonal antibodies are immobilized at the second location. Anti-ECF monoclonal antibodies recognizing different epitopes can be used in both locations.

The apparatus may further include means for directing the sample containing the fluid to the selected location in the support. In a further embodiment, a blocking solution, as described herein, may be added after the sample is placed in the support.

In this application, reference is made to several publications. The descriptions of these publications in their entirety are incorporated herein by reference for a more complete description of the state of the art to which this invention pertains.

EXAMPLES Example 1. Purification of ECF The serum was collected from the cattle at 12-48 hours after fertilization in volumes greater than 250 ml and frozen until the pregnancy was confirmed in 45 days. When the serum was approved for the extraction of ECF, by confirming the pregnancy by other means, they were thawed at room temperature. Equal volumes of a diluted perchloric acid solution (70% perchloric acid diluted in a ratio of 1 ml per 50 ml of distilled water) and the serum were mixed using a magnetic stirrer. The mixture was kept in an ice bath with constant stirring for 1 hour. Subsequently the mixture was centrifuged for 30 minutes at 2000 rpm using a refrigerant centrifuge. The resulting cream was dialysed for 72 hours against distilled water, then the presence of ECF was verified by immunological documentation.

The dialyzed cream, stabilized at pH 7.4 with sodium phosphates, containing ECF was further purified using a highly purified cellulose powder containing diethylaminoethyl (DEAE) exchange groups equilibrated in 0.025 M sodium phosphate stabilizer at pH 7.4. The non-absorbed fraction of the DEAE powder was collected as Fraction A. The material in the cream that bound to the DEAE column was eluted using 0.05 M Sodium Phosphate, 0.9% sodium chloride pH 7.4 and was collected as Fraction B. The fractions A and B each were dialyzed against distilled water.

Fraction A subsequently was further purified by passing it on a Sepharose 4B with HCL Tris 0.05 M pH 7.4 and two maximum readings, labeled as Fraction A1 and Fraction A2, were collected and recombined to reconstitute Fraction A.

The two ECF fractions, A and B (reconstituted as described) and a combination of equal milligrams of Fractions A and B, were each diluted 1 milligram per ml in normal saline. These three preparations were used to immunize three goats labeled separately for the production of antibodies. The antiserum of the three goats was tested against each of the three immunization preparations. For example, Goat No. 20, which was immunized with the combination of Fraction A and Fraction B, showed strong immunological reactivity against the three immunization antigens.

Example 2. Production of Anti-ECF Polyclonal Antibodies The labeled goats were immunized with the purified ECF fractions A and B of Example 1, which were combined and diluted to 1 mg / ml.

Eight primary injections of antigen were placed on each goat using Freund's complete adjuvant. A typical immunization antigen preparation contained 20 ml of Fraction A and fraction B reconstituted (both prepared as described in Example 1) in 1 mg / ml and 20 milliliters of Freund's Complete Adjuvant. Two (2) millimeters of this preparation were removed and homogenized to less than 1 millimeter which was subsequently injected into the goat muscle. The injections were applied twice a week, with three days of separation. At eight days after the eighth injection, the goats bled and the antibodies were extracted from the blood. Antibodies were analyzed to determine activity against purified ECF. Animals that tested positive were subsequently given monthly pressure boosting injections and were bled eight days after the booster injection to provide a constant source of antiserum.

To increase the specificity of the antiserum as it was produced, each collection of serum was absorbed with normal human serum collected from unimmunized cattle until the lines were not visible in the Ouchterlony Gel Diffusion studies. This pre-absorbed antiserum was further purified using sodium sulfate fraction. The resulting antibody preparations were subsequently used for the development of a haemagglutination inhibition test and an enzyme immuno test. In addition the antibodies were also purified using a chromatographic column of Precept A (Bioprocessing Ltd. Durham, United Kingdom), eluted with a phosphate-buffered saline solution at a pH of 7.4 and these antibodies were used in a penetration band test.

Example 3. Production of Anti-ECF Monoclonal Antibodies Balb C mice were used for immunization using the immunization preparations described in Example 2. The same immunization schedule that was used for the goats was followed for the mice, except that two following days from the last injection, fusion of mouse spleen cells with SPS / 0-Ag melanoma cells (available from the American Type Culture Collection) was performed using 30% polyethylene glycol in RPMI 1640 medium. Standard maintenance was followed of the hybridoma cells in hypoxanthine, aminopterin and thymidine (HAT) containing the medium (Goding, 1983 and Harlow and Lane, 1988). The antibody producing cells with the strongest solution concentration were identified using hemagglutination procedures with red blood cells that were covered with ECF, prepared following standard procedures known in the art.

The selected hybridoma cells were propagated for the production of monoclonal antibodies. Cloning a specific hybridoma cell line by limiting the dilution in the fluid phase and semisolid agarose techniques. The anti-ECF antibody producing clones was maintained and grown in volume using HAT containing DMEM (Dulbecco's modified Eagle's medium), using protocols known in the art (Harlow and Lane, 1988).

An anti-ECF monoclonal antibody selected by said methods was bombarded with isotopes and documented to be an IgA. Reacted with several preparations of ECF antigens by Western blot analysis. This monoclonal antibody was coupled to colloidal gold using methods known in the art (eg, Julián Beesley, Colloidal Gold, Oxford Press, 1989).

Example 4. Construction of a Test Equipment for the Determination of Conception State A kit was constructed using the anti-ECF polyclonal antibodies described in Example 2 linked to a 4.5 x 0.5 centimeter (cm) band of Whatman nitrocellulose membrane. 5 microns. The anti-ECF monoclonal antibodies, as described in Example 3, were coupled to the colloidal gold to form a conjugate and deposited on a 2.5 x 0.5 cm pad of Whatman OF-75 material.

The equipment was mounted as a lateral flow device by placing the two bands containing the end-to-end antibodies as a 7 cm band, with the animal's test sample to be introduced to the FO75 pad, ie the "sample end" . A second pad, made of CHO-17 glass fiber material, was placed at the other end (ie, the end does not show) to assist in the passage of fluid from the sample end and through the nitrocellulose membrane band. Anti-ECF polyclonal antibodies were bound to the membrane in a "Test" band (approximately 0.1 cm wide) located approximately 3.4 cm from the sample end of the band. An IgG goat control antibody (Sigma, St. Louis, MO) was ligated into a "Control" band of similar size located approximately 0.5 cm from the anti-ECF polyclonal antibodies at the end of this distal band of the sample end. .

Example 5. Performing the ECF Test for "Open" cattle A serum sample from a beef being tested was introduced as a drop in the band equipment of Example 4 at the sample end of the band, approximately 1 cm from the end of the band. Approximately 2-8 drops of the blocking stabilizer were added directly into the supepor portion of the serum sample and the liquid was allowed to pass along the band to the two areas bound with antibodies. The presence of a single line in the band (which would be located in the control band) indicates that the beef is "open", that is, the beef has not been conceived. The presence of two lines in the band, located in each of the control and test bands, indicates that a res has conceived.

The test was performed on 153 cattle that had been artificially inseminated and the results of the ECF test were compared to the results of veterinary tact. Of the 65 animals that through veterinary touch were shown to be pregnant, 53 were positive in the ECF test. Of the 89 determined animals that were not pregnant according to touch, 45 were negative in the ECF test.

Example 6. Test for ECF in Humans Samples were collected from patients, who were artificially inseminated. The samples were analyzed to demonstrate the presence of ECF using a urease-anti-ECF conjugate. The following data was collected: Sample Time after insemination (in days) Patient No. 0.25 2.0o 6 a.0o 12.0 Pregnant? 1 .358 .120 .100 .100 not 2 .114 .095 .094 .091 not 3 .103 .099 .093 .098 no. 4 .103 .096 .100 .095 no. 5 .070 .079 .052 .088 no 6 .104 .096 .081 .108 no 7 .301 .159 .105 .111 no 8 .104 .106 .094 .101 no 9 .102 .098 .092 .058 no 10 .075 .085 .091 .050 no 11 .075 .082 .078 .087 not 12 .073 .076 .081 .096 not 13 .079 .081 .078 .078 not 14 1,657 1,691 1,674 1,557 if 15 1,660 1,690 1,708 1,577 yes 16,087,070,087. 088 No 17 .074 .078 .075 .077 No 18 .085 .077 .074 .074 No 19 .087 .081 .075 .030 No 20 .082 .077 .079 .096 No 21 .084 .079 .074. 075 not 22 .083 .078 .078 .077 if * 23 .087 .088 .090 .078 not 24 .077 .074 .078 .080 not Patient No. 15 aborted at 3 months. Patient No. 22 aborted at 6 weeks.

Although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that said details should be limiting of the scope of the invention. They are duly included in the accompanying claims.

Claims (20)

1. An isolated antibody that binds specifically to the initial purified design factor.
2. An isolated antibody according to claim 1, which has a molecular weight of about 200,000.
3. An isolated antibody according to claim 2, wherein the initial design factor is of a species selected from the group consisting of cattle, cats, dogs, horses, humans, sheep and pigs.
4. An antibody according to any of claims 1, 2 or 3, wherein said antibody is selected from the group consisting of polyclonal, monoclonal, humanized, fully human or chimeric antibodies.
5. A method for detecting conception in an animal that comprises detecting the presence of the initial design factor in a body fluid of the animal.
6. The method according to claim 5, wherein the body fluid is serum.
7. The method according to claim 5, wherein the body fluid is urine.
8. The method according to claim 5, wherein the body fluid is milk.
9. A method to determine the absence of conception in a res in the first twelve hours of gestation, which includes the determination of the presence or absence of the initial conception factor, the absence of the initial conception factor indicates the absence of conception.
10. A method to determine the absence of conception in an animal in the first twenty-four hours of gestation, which includes the determination of the presence or absence of the initial conception factor, the absence of the initial conception factor indicates the absence of conception.
11. A method for detecting the initial conception factor in an animal comprising the steps of: a) collect a sample of the animal; b) contact the sample with the anti-antibodies (initial conception factor) under conditions where the antibodies can bind the protein of the initial conception factor present in the sample and c) detect the antibody of the initial design factor complexes.
12. The method according to claim 11, wherein the sample is selected from the group consisting of serum, urine and milk.
13. The method according to claim 11, wherein the initial anti-factor of design antibody is conjugated to a detectable part and the antibody complex of the initial design factor is detected by addition of a substrate or inducer and the changes are monitored. detectable in the receptacle.
14. The method according to claim 13, wherein the anti-(initial design factor) antibodies are conjugated to a selected portion of the group consisting of alkaline phosphatase, equine peroxidase, colloidal gold and urease.
15. An apparatus for detecting the initial design factor in a fluid containing a sample of a subject comprising: a) a body portion and b) a support having therein, an antibody for an initial design factor in contact with the body portion.
16. 16. The apparatus according to claim 15, wherein the support comprises a material that passes a fluid.
17. 17. The apparatus according to claim 15, wherein the antibody is conjugated to a detectable part.
18. 18. The apparatus according to claim 15, wherein the substrate has a polyclonal and anti-monoclonal antibody (initial design factor) therein.
19. 19. The apparatus according to claim 18, wherein the anti-monoclonal and polyclonal antibodies (initial design factor) are spatially separated in the support.
20. 20. The apparatus according to claim 18, wherein the polyclonal antibody is located in a band, wherein the band is substantially perpendicular to the longitudinal axis of the support. The apparatus according to claim 18, further comprises means in the body portion for directing a fluid to the support. The apparatus according to claim 21, wherein the means in the body portion for directing a fluid to the support directs the fluid to a location on the support where the monoclonal antibody is located, where the sample contacts the monoclonal antibody and the support passes the monoclonal antibody and the fluid comes into contact with the band containing the polyclonal antibody.