WO2004095033A2

WO2004095033A2 - Compositions and methods for accurate early pregnancy diagnosis in ungulates

Info

Publication number: WO2004095033A2
Application number: PCT/US2004/011456
Authority: WO
Inventors: Nagappan Mathialagan
Original assignee: Mosanto Technology Llc
Priority date: 2003-04-17
Filing date: 2004-04-14
Publication date: 2004-11-04
Also published as: WO2004095033A3

Abstract

This invention provides improved methods for early detection of ungulate (e.g., bovine) pregnancy, and novel antibodies useful in such assays. The antibodies are generated by using specific amino acid sequences within ubiquitin cross-reactive protein (UCRP) as immunogens. Elevated levels of UCRP proteins, when detected in a sample (e.g., bodily fluid) of the animal from days 15-30, and especially days 15-28 after insemination, indicate that the animal is pregnant. The invention can be employed to increase efficiency of commercial farm animal breeding programs.

Description

COMPOSITIONS AND METHODS FOR ACCURATE EARLY PREGNANCY

DIAGNOSIS IN UNGULATES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application Serial No. 60/463,780, filed April 17, 2003, U.S. Provisional Patent Application Serial No. 60/463,830, filed April 17, 2003, and U.S. Provisional Patent Application Serial No. 60/465,611, filed April 24, 2003, each of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

[0002] This invention relates generally to the fields of veterinary medicine, reproductive biology and diagnostics, and more specifically, improved methods for early stage pregnancy detection in ungulates (hoofed animals), including ruminants such as bovines, e.g., dairy and beef cattle, and ovines.

II. Related Art

[0003] Pregnancy diagnosis is an important component of sound reproductive management, particularly in the dairy industry (Oltenacu et al, 1990), where a high proportion of artificial inseminations fail (Streenan and Diskin, 1986). A reliable yet simple pregnancy test for cattle has long been sought. Several procedures are available, including a milk progesterone assay (Oltenacu et al, 1990; Markusfeld etal, 1990), estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al., 1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal et al., 1992; Cameron and Malmo, 1993), and blood tests for pregnancy-specific antigens. Of these, a progesterone milk assay is said to be the most cost-effective for the producer (Oltenacu et al., 1990; Markusfeld etal, 1990). Next best is rectal palpation performed at day 50 post- insemination (Oltenacu et al, 1990).

[0004] Even though the prior procedures for pregnancy diagnosis are potentially useful, all have fallen short of expectations in terms of their practical, on-farm use. For example, measurements of milk or serum progesterone around day 18-22 yield high rates of false positives (Oltenacu et al, 1990; Markusfeld et al, 1990). The presence of estrone sulfate in urine or serum provides another test, but is only useful after day 100 as concentrations rise (Holdsworth et al, 1982; Warnick et al, 1995). [0005] In general, artificial insemination is successful less than 50% of the time, and the producer must either rely on overt signs of return to estrus (that are easily missed) or delay rebreeding until pregnancy failure is confirmed by one of the methods described above. Such delays are very costly and a major economic loss to the industry. There is, therefore, a need for an early pregnancy test that is economical, convenient, noninvasive, and provides improved accuracy.

[0006] In a related field, it has been discovered that, in bovines, one of the dominant proteins released by the conceptus (at about 13 to 20 of pregnancy) is interferon-tau (IFN-tau), originally called trophoblastin or trophoblast protein- 1 (Hansen et al, 1999). After treating endometrial explants from day 18 pregnant cows Naivar et al. (Naivar et al, 1995) identified a 17kDa uterine protein that was released into the medium in response to both recombinant IFN-tau and recombinant IFN-alpha. The protein was found to be similar in size to human IFN-stimulated gene product- 15 (also called huUCRP) and to be immunoreactive with anti-serum against ubiquitin. This protein has, therefore, been termed bovine ubiquitin cross-reactive protein ("UCRP") (Austin et al. 1996). Subsequently, the amino acid sequence (Accession No. AAB57687) and corresponding nucleotide sequence (Accession No. U96014) were determined (Austin et al. 1996 and 1997). All accession numbers referred to herein are found in GenBank. The bovine UCRP amino acid sequence is listed herein as SEQ ID NO:34 with the corresponding nucleotide sequence listed for reference as SEQ ID NO:35. All UCRP numbering referred to herein is based upon the amino acid sequence numbering from SEQ ID NO:34 (Accession No. AAB57687, Austin et al. 1997).

[0007] Bovine UCRP (also known as bovine IFN-stimulated gene product- 17, or ISG17) is produced by uterine endometrium in response to IFN-tau that is secreted by bovine conceptus from day 13 to day 20 of pregnancy. During the pre-implantation period, IFN-tau secreted by the trophoblast layer of the conceptus functions as a maternal signal denoting recognition of pregnancy in ruminants.

[0008] Molecular cloning and sequencing of IFN-tau has shown that it belongs to Type I IFN class of proteins (IFN-alpha and IFN-beta). IFN alpha and IFN beta also interact with Type I receptors on the cellular membranes and utilize similar signal transduction pathways. Receptors for IFN-tau are present on the endometrial epithelial cells of the uterus. Sequencing and binding studies have shown that these IFN-tau receptors belong to IFN Type I receptor class. [0009] Hansen et al, 1997 reports the pregnancy specific secretion of ISG17 by the endometrium during days 15 to 26 of pregnancy in response to conceptus-derived IFN-tau. In a related study, it has been reported that the ovine uterus also produces UCRP in response to IFN-tau treatment (Johnson et al, 1999). Finally, it has been suggested that ISG17 may be released into peripheral circulation via the histotroph (also known as uterine milk) that nourishes the embryo until the placenta forms. (Austin et al, 1996; Pru et al, 2000). [0010] The use of antibodies for determining pregnancy in an ungulate (e.g., a bovine) by immunologically detecting elevated levels of full-length (native) UCRP in a sample from the animal is disclosed in U.S. Provisional Patent Application 60/435,540 filed December 19, 2002, now pending Serial No. PCT/US03/40192, the disclosures of which are incorporated herein by reference. To facilitate the use of such a method for pregnancy determination, it is desirable to identify amino acid epitopes that contain antigenic amino acid sequences or fragments of full- length UCRP for use as immunogens for the production of antibodies useful in that method, without the need to obtain the full-length UCRP (by purification or production, e.g., using recombinant DNA) in the quantities that would be needed to produce such antibodies.

SUMMARY OF THE INVENTION [0011] This invention provides methods for the early detection of pregnancy in livestock such as ungulates (e.g., hoofed animals). In one aspect of the invention, methods are provided for the early detection of pregnancy in ruminants, e.g., a bovine or ovine animal suspected of being pregnant, comprising obtaining a sample from the animal, measuring the level of UCRP in the sample with an antibody prepared by any of the methods described herein, wherein elevated levels of UCRP indicate that the animal is pregnant. The sample may be of any biological material including saliva, urine, or milk, and is preferably whole blood, serum, or plasma. In certain embodiments of the invention, the sample may be obtained from the animal from about day 15 to 30 post-insemination, preferably from day 15 to 28, more desirably from day 15 to 25 or,from day 15 to about 20 post-insemination, including most advantageously from about day 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 post-insemination.

[0012] One aspect of the present invention includes methods for predicting antigenic sequences of an ubiquitin cross-reactive protein (UCRP) comprising: (a) selecting an analysis program that predicts antigenic amino acid sequences; (b) inputting the UCRP amino acid sequence into the analysis program; (c) obtaining the predicted antigenic amino acid sequences; (d) aligning the UCRP sequences with protein sequences for which X-ray crystal structure data is known and that share at least 80% sequence identity with said UCRP protein to obtain predicted X-ray crystal structure data for UCRP; (e) comparing the predicted antigenic amino acid sequences of the UCRP protein obtained in step (c) to the three dimensional X-ray crystal structure data of step (d); and (f) identifying antigenic UCRP amino acid sequences based upon the comparative data from step (e). In step (d) it is contemplated that the UCRP protein and aligned protein sequences will share at least 80% sequence identity; however, it is also dersirable that the (d) proteins share even greater sequence identiy, including from 80-100% identity, preferably at least 85% identity, or more preferably at least 90% identity. It is also contemplated that the proteins aligned in step (d) share 90-95% identity, or even more preferably, that they share at least 95% identity. It is also contemplated that the shared identity be exhibited in regions or domains of the protein (contrasted to the entire protein sequence), ranging from at least three amino acids, and typically the domains or regions may be from three to twenty amino acid residues in length, or greater. The method also includes identifying antigenic UCRP amino acid sequences that are within at least one of six antigenic regions comprising:

I. SEQ ID NO:28, (UCRP amino acids 4-11);

II. SEQ ID NO:29, (UCRP amino acids 19-33);

III. SEQ ID NO:30, (UCRP amino acids 44-55);

IV. SEQ ID NO:31, (UCRP amino acids 83-97);

V. SEQ ID NO:32, (UCRP amino acids 97-116); or

VI. SEQ ID NO:33, (UCRP amino acids 124-143).

[0013] The method further includes identifying antigenic UCRP amino acid sequences that are selected from the group consisting of: SEQ ID NO:l, (GDLTV, amino acids 3-7 of UCRP); SEQ ID O:2, ( LGGQEI, amino acids 10-15 of UCRP); SEQ ID O:3, (LRDSM, amino acids 19-23 of UCRP); SEQ ID NO:4, (RDSMT, amino acids 20-24 of UCRP); SEQ ID NO:5, (KQF, amino acids 29-31 of UCRP); SEQ ID NO:6, (PAFQQR, amino acids 39-44 of UCRP); SEQ ID NO:7, (QGLRAG, amino acids 63-68 of UCRP); SEQ ID NO:8, (LVVQNC, amino acids 73-78 of UCRP); SEQ ID NO: 9, (ISILVR, amino acids 79-84 of UCRP); SEQ ID NO: 10, (RNDKGR, amino acids 84-89 of UCRP); SEQ ID NO: 11, (DKGRSS, amino acids 86-91 of UCRP); SEQ ID NO: 12, (KGRSS, amino acids 87-91 of UCRP); SEQ ID NO: 13, (VQLKQ, amino acids 95- 99 of UCRP); SEQ ID NO:14, (QADQFW, amino acids 115-120 of UCRP); SEQ ID NO:15, (EEYG, amino acids 135-138 of UCRP); SEQ ID NO:16, (YGLMK, amino acids 137-141 of UCRP); SEQ ID NO:17, (MKG, amino acids 140-142 of UCRP); SEQ ID NO:18, (NLRLRG, amino acids 148-153 of UCRP); SEQ ID NO:19, (LAH, amino acids 45-47 of UCRP); SEQ ID NO:20, (DSRE, amino acids 49-52 of UCRP); SEQ ID NO:21, (QTVA, amino acids 99-102 of UCRP); SEQ ID NO:22, (KQQ, amino acids 105-107 of UCRP); SEQ ID NO:23, (QKER, amino acids 110-113 of UCRP); SEQ ID NO:24, (MDDEH, amino acids 128-132 of UCRP); SEQ ID NO:25, (VSELK, amino acids 25-29 of UCRP); SEQ ID NO:26, (PEYVQ, amino acids 92-97 of UCRP); SEQ ID NO:27, EGRPMD, amino acids 124-129 of UCRP); and combinations thereof.

[0014] Another aspect of the invention includes antibodies generated by using at least one of the antigenic UCRP amino acid sequences identified by the above method for predicting antigenic sequences of UCRP, as an immunogen. These antibodies include those that are generated using antigenic UCRP amino acid sequences that are within at least one of six antigenic regions comprising:

I. SEQ ID NO-.28, (UCRP amino acids 4-11);

II. SEQ ID NO:29, (UCRP amino acids 19-33);

III. SEQ ID NO:30, (UCRP amino acids 44-55);

IV. SEQ ID NO:31, (UCRP amino acids 83-97);

V. SEQ ID NO:32, (UCRP amino acids 97-116); or

VI. SEQ ID NO:33, (UCRP amino acids 124-143).

[0015] The antibodies that are useful in this invention include those that have been generated by using one or more of the following amino acid sequences as an immunogen, preferably the antigenic sequence is similar to epitopes or antigenic regions from within full-length ungulate/bovine UCRP, and preferably the UCRP antigenic sequences contain a surface portion of a three-dimensional UCRP protein conformation as it would normally be found in the animal including the following amino acid sequences: SEQ ID NO: 1, (GDLTV, amino acids 3-7 of UCRP);

SEQ ID NO: 2, (LGGQEI, amino acids 10-15 of UCRP);

SEQ ID NO: 3, (LRDSM, amino acids 19-23 of UCRP);

SEQ ID NO: 4, (RDSMT, amino acids 20-24 of UCRP);

SEQ ID NO: 5, (KQF, amino acids 29-31 of UCRP);

SEQ ID NO: 6, (PAFQQR, amino acids 39-44 of UCRP);

SEQ ID NO: 7, (QGLRAG, amino acids 63-68 of UCRP);

SEQ ID NO: 8, (LVVQNC, amino acids 73-78 of UCRP);

SEQ ID NO: 9, (ISILVR, amino acids 79-84 of UCRP);

SEQ ID NO: 10, (RNDKGR, amino acids 84-89 of UCRP);

SEQ ID NO: 11, (DKGRSS, amino acids 86-91 of UCRP);

SEQ ID NO: 12, (KGRSS, amino acids 87-91 of UCRP);

SEQ ID NO: 13, (VQLKQ, amino acids 95-99 of UCRP);

SEQ ID NO:14, (QADQFW, amino acids 115-120 of UCRP);

SEQ ID NO: 15, (EEYG, amino acids 135-138 of UCRP);

SEQ ID NO:16, (YGLMK, amino acids 137-141 of UCRP);

SEQ ID NO: 17, (MKG, amino acids 140-142 of UCRP);

SEQ ID NO:18, (NLRLRG, amino acids 148-153 of UCRP);

SEQ ID NO: 19, (LAH, amino acids 45-47 of UCRP);

SEQ ID NO:20, (DSRE, amino acids 49-52 of UCRP);

SEQ ID NO:21, (QTVA, amino acids 99-102 of UCRP);

SEQ ID NO:22, (KQQ, amino acids 105-107 of UCRP);

SEQ ID NO:23, (QKER, amino acids 110-113 of UCRP);

SEQ ID NO:24, (MDDEH, amino acids 128-132 of UCRP);

SEQ ID NO:25, (VSELK, amino acids 25-29 of UCRP);

SEQ ID NO:26, (PEYVQ, amino acids 92-97 of UCRP);

SEQ ID NO:27, (EGRPMD, amino acids 124-129 of UCRP); and combinations thereof. The terms amino acid sequences, residues, fragments, regions, and epitopes are used throughout the description to identify certain desired amino acid sequences that may exhibit any one or combination of desired characteristics including being antigenic, surface exposed, and exhibiting a useful conformation that is found in the native UCRP protein. The terms amino acid sequences, residues, and fragments are used synonymously.

[0016] Antibodies useful in this invention can be generated by using more than one of the above- listed amino acid sequences as an immunogen. Each of such antibodies can, and commonly does, bind to or immunoreacts with amino acid sequences within full-length UCRP that are longer than but contain one or more of the above-listed sequences. To illustrate, but not by way of limitation, antibodies useful in this invention may bind to or immunoreact with the following sequences: (a) SEQ ID NO:5 and an additional L (amino acid 28 of UCRP) or EL (amino acids 27-28 of UCRP) and/or an additional IA (amino acids 32-33 of UCRP); (b) SEQ ID NO: 15 and an additional L (amino acid 134 of UCRP) and/or an additional L (amino acid 139 of UCRP); (c) SEQ ID NO:17 and an additional C (amino acid 143 of UCRP); (d) SEQ ID NO: 19 and an additional R (amino acid 44 of UCRP); SEQ ID NO: 21 and an additional K (amino acid 98 of UCRP) or LK (amino acids 97-98 of UCRP) and/or an additional E (amino acid 103 of UCRP); SEQ ID NO:22 and an additional V (amino acid 108 of UCRP); and/or SEQ ID NO:23 and an additional V (amino acid 114 of UCRP).

[0017] Antibodies useful in this invention can be generated using more than one of the above- listed amino acid sequences as the immunogen. For example, antibodies useful in specific embodiments of the invention are generated by using at least two (in some cases preferably three) of the following combinations of such amino acid sequences as the immunogen for generating an antibody: SEQ ID NOs: 1, 3 and 7; SEQ ID NOs: 2, 6 and 8; SEQ ID NOs: 9, 13 and 16; and SEQ ID NOs: 12, 14 and 18. Each of such antibodies can, and commonly do bind, alternatively or also, to amino acid sequences within full-length UCRP that are longer than but contain one or more of the above-listed sequences.

[0018] In one embodiment of the invention, UCRP is present in early pregnancy and, at about two months (preferably about one month) post-partum, is essentially absent (undetectable by methods commonly available at this time) or at least not present above a "background" or "baseline" level that can be measured and established as a reference point against which the UCRP levels that are detected in accordance with this invention can be evaluated as being "elevated" or "not elevated."

[0019] Antibodies useful in this invention can include monoclonal antibodies or polyclonal antisera. The immunologic detection may be carried out using any suitable techniques, including ELISA, RIA, and Western blot. The ELISA may comprise a sandwich ELISA comprising binding of UCRP to a first antibody preparation fixed to a substrate and a second antibody preparation labeled with an enzyme. In one embodiment, the enzyme is alkaline phosphatase or horseradish peroxidase. In certain embodiments of the invention, an elevated level of UCRP is any amount greater than about 0.5 ng/ml of serum. Typically, elevated levels range from about 0.5 ng/ml to about 10 ng/ml of serum, and more commonly from about 1 ng/ml to about 4 ng/ml. However, any UCRP level that is above about 0.5 ng/ml is considered to be elevated above baseline, that is elevated above the level of UCRP in a non-pregnant ungulate animal. [0020] In yet another aspect, the invention provides a method of making a breeding decision for a bovine animal comprising: using at least one of the antibodies of this invention to determine whether the level of UCRP in a sample from an animal suspected of being pregnant is elevated, wherein (i) elevated levels of UCRP indicate that the animal is pregnant, and no further steps need be taken; and (ii) non-elevated levels of UCRP indicate that the bovine animal is not pregnant, and should be treated to impregnate in a timely manner, e.g., injected with gonadotropin-releasing hormone (GnRH), and about seven days later, injected with prostaglandin F_2α (PGF), followed by re-insemination. The method may comprise, about 48 hours after the PGF injection and before re-insemination, administering a second injection of GnRH.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0021] This invention overcomes limitations of the prior art by enabling the more convenient preparation of antibodies useful in methods for reliably determining early pregnancy. An accurate yet simple pregnancy test for cattle has long been sought. Previously, commercially available tests have either not allowed early detection of pregnancy or have suffered from a high incidence of false positive or false negative results. The prior tests, although potentially useful, have fallen short of expectations in terms of their practical, on-farm value and use. [0022] Antibodies useful in this invention can be prepared using any of various techniques well known in the art, and by using UCRP sequences or fragments that have three-dimensional protein conformations suitable for generating antibodies that faithfully react with full-length (native) UCRP as immunogens, as described herein. Typically, such fragments can be synthesized more economically than purifying enough full-length UCRP to prepare the antibodies, or by synthesizing full-length UCRP, e.g., by the use of recombinant DNA, for that purpose. However, if desired, these antibodies can be raised against such full-length UCRP. I. Identification of antigenic fragments of UCRP.

A. Identification and selection of UCRP fragments for generating antibodies.

[0023] The desired, antigenic sequences of bovine UCRP (also referred to as ISG17) were identified by interpreting and analyzing the results obtained from various sequence analysis algorithms and by conserved protein domain analysis.

[0024] The UCRP sequence analysis was performed using the Protean™ component of the DNA Star™ sequence analysis package. The Protean™ program provides a series of software packages that determine the structural features of input protein sequences. [0025] The algorithm used for the analysis predicts the secondary structural features such as alpha-helix and beta sheet, hydrophobicity and surface probability of amino acid residues. Four different algorithms from the Protean™ program were used to generate data for predicting antigenic sites of the UCRP protein, including the Sette MHC motifs (Sette et al. 1989), the AMPHI analysis (Margalit et al. 1987), the Rothbard-Taylor analysis (Rothbard and Taylor, 1988), and the Jameson- Wolf analysis (Jameson and Wolf, 1988). The Sette MHC motif analysis predicts peptide antigenic sites that interact with mouse MHC II haplotype d proteins. The AMPHI analysis predicts immunodominant helper-T-lymphocyte antigenic sites from primary sequence data. The Rothbard-Taylor analysis locates potential T-lymphocyte antigenic determinants that contain a common sequence motif. Finally, the Jameson- Wolf analysis predicts potential antigenic determinants by combining existing methods for protein structural predictions including hydrophobicity Hopp-Woods values (Hopp and Woods, 1981), (Hopp- Woods, H [<2>0.5<-l>-0.5<-2>]), surface probability values of Emini (Emini et al. 1985), (Emini, S [<1>1.0<0>]), flexibility values of Karplus-Schultz (Karplus et al. 1985), (Karplus- Schultz, F [<1>1.0<0>]), secondary structure predictions of Chou and Fasman (1978), (Chou- Fasman, CF [T=2, t=l, 0]) and Garnier-Robson (Garnier et al. 1978), (Garnier-Robson, GR [T=2, t=l, 0]) using the following formula: A= 0.3H+ 0.15S+0.15F+0.2CF+0.2GR. [0026] The bovine UCRP amino acid sequence (SEQ ID NO:34) was analyzed with the Protean™ program, which identified six regions, each containing numerous potentially antigenic sequences and/or fragments of UCRP. [0027] The Sette analysis predicted that the following sequences are likely to be antigenic:

UCRP amino acid residues 28-33; LKQFIA (6 residues);

UCRP amino acid residues 62-70; LQGLRAGST (9 residues);

UCRP amino acid residues 84-89; RNDKGR (6 residues). [0028] The AMPHI analysis predicted that the following sequences have a high amphipathic index indicating that they may be antigenic:

UCRP amino acid residues 27-31; ELKQF (5 residues);

UCRP amino acid residues 45-47; LAH (3 residues);

UCRP amino acid residues 77-82; NCISIL (6 residues);

UCRP amino acid residues 99-103; QTVAE (5 residues);

UCRP amino acid residues 134-143; LEEYGLMKGC (10 residues). [0029] The Rothbard-Taylor method utilizing the T-cell motif analysis identified the following sequences:

UCRP amino acid residues 4-11; DLTVKMLG (8 residues);

UCRP amino acid residues 44-47; RLAH (4 residues);

UCRP amino acid residues 52-55; EVLQ (4 residues);

UCRP amino acid residues 138-141; GLMK (4 residues). [0030] The Jameson- Wolf analysis predicted the following sequences:

UCRP amino acid residues 19-29; LRDSMTVSELK (11 residues);

UCRP amino acid residues 46-55; AHLDSREVLQ (10 residues);

UCRP amino acid residues 83-97; VRNDKGRSSPYEVQL (15 residues);

UCRP amino acid residues 100-116; TVAELKQQVCQKERVQA (17 residues);

UCRP amino acid residues 124-139; EGRPMDDEHPLEEYGL (16 residues). [0031] Using the combined data generated by the four Protean™ predictions as shown above, the following six UCRP sequence regions, were selected as being potentially antigenic residues or fragments: [0032] Region I. SEQ ID NO: 28, which consists of UCRP amino acid residues 4-11:

DLTVKMLG (predicted by Rothbard-Taylor analysis). [0033] Region II. SEQ ID NO:29, which consists of UCRP amino acid residues 19-33: LRDSMTVSELKQFIA (residues 19-29 were predicted by Jameson- Wolf; the overlapping sequence motifs of residues 28-33 and 27-31, were predicted by Sette and AMPHI, respectively); [0034] Region III.SEQ ID NO: 30, which consists of UCRP amino acid residues 44-55:

RLAHLDSREVLQ (residues 46-55 were predicted by Jameson-Wolf; residues 45-47 were predicted by AMPHI; and residues 44-47 were predicted by Rothbard-Taylor); [0035] Region IV. SEQ ID NO: 31, which consists of UCRP amino acid residues 83-97:

VRNDKGRSSPYEVQL (predicted by Jameson-Wolf; residues 84-89 were also predicted by Sette); [0036] Region V. SEQ ID NO: 32, which consists of UCRP amino acid residues 97-116:

LKQTVAELKQQVCQKERVQA (residues 100-116 were predicted by

Jameson- Wolf; residues 97-102 were predicted by Sette; and residues 99-103 were predicted by AMPHI; [0037] Region VI. SEQ ID NO:33, which consists of UCRP amino acid residues 124-143:

EGRPMDDEHPLEEYGLMKGC (residues 124-139 were predicted by

Jameson-Wolf; residues 134-143 were predicted by AMPHI; and residues

134-143 were predicted by Rothbard-Taylor. [0038] An additional type of analysis, the conserved domain database (CDD) analysis system was used to predict 'conformation' epitope sequences based on X-ray crystallographic data available from related family members of UCRP/ISG17. The NCBI (National Center for Biotechnology Information) website is among the sources offering this analysis program (listed as CDD search, currently version 1.65; Marchler-Bauer et al. 2003 and 2002). [0039] The CDD search runs reverse-position specific BLAST (RPS-BLAST) utilizing the target protein sequence, in this case UCRP. The search is performed and aligns the target protein sequence with the database of protein domains that are conserved in molecular evolution.

[0040] The CDD search with the UCRP sequence identified a number of proteins that shared conserved sequence domains with the UCRP sequence, and for which X-ray crystal structure data is available. These proteins included Sumo-1 (Accession No. 1A5R, GI 3892023); Nedd8 (Accession No. Q15843), and Smt3 (Accession No. Q12306). The X-ray crystal structures for these three proteins have been solved and are available on the NCBI web site. [0041] The conserved domain database search with the UCRP protein sequences identified six hits, with the best alignment to the conserved domain being "pfam00240.7," ubiquitin, and PSSM-Id:7525. The best alignments with UCRP in the conserved domain database are: Smt3 (Accession No. Q12306); Sumo-1 (Accession No. 1A5R, GI 3892023); and Nedd8 (Accession No. Q15843).

[0042] The UCRP amino acid residues 5-78 and 79-154 exhibited 97% and 100% alignment with the sequence of Smt3, suggesting that UCRP shares at least two domains with the Smt3 protein. Each of these apparently shared domains consists of about 75 amino acid residues and is referred to as a "conformational specific region or epitope".

[0043] Since the X-ray crystal structure of Smt3 has been solved and is in the NCBI database, the conserved domain database analysis program can be used to effectively substitute the amino acid residues of a desired protein, in this case, UCRP, into the crystal coordinates of the known structure, such as Smt3 in the present example. The conservation of the sequences between the query protein (UCRP) and the known protein structure in the conserved domain database hit (Smt3) indicates that the query protein's and the known protein's crystal structures should be identical. This prediction or assumption has been shown to be true in numerous examples of proteins that share at least two conserved domains, such as ovalbumin and trypsin inhibitor. This CDD analysis program also enables one to view the results of such a prediction by providing an aligned view of the conserved domains using the C3nD program. These structural illustrations allow for the identification of amino acid residues from the query sequence that form folds to generate specific conformations.

[0044] Utilizing this CDD analysis for UCRP, some specific short stretches of sequences that are not contiguous with regard to the linear amino acid sequence, have been identified that appear to form conformational regions or epitopes. Since these conformation regions or epitopes are likely to contribute to the unique conformation and binding properties of UCRP, these conformation regions or epitopes are likely to be recognized by conformational specific antibodies. Such amino acid residues, fragments, or combinations within these conformational regions or epitopes will also be useful for using as immunogens for generating desired antibodies.

[0045] Summarizing the conserved domain analysis results shows that there are four conformational specific domains or epitopes, each comprising three noncontiguous amino acid segments that could be identified based upon the tertiary (3-D) X-ray structure prediction comparing UCRP with Smt3:

Conformation specific domain 1: SEQ ID NO:l (UCRP amino acids 3-7, GDLTV); SEQ ID NO:3 (UCRP amino acids 19-23, LRDSM); and SEQ ID NO: 7 (UCRP amino acids 63-68, QGLRAG); Conformation specific domain 2: SEQ ID NO: 2 (UCRP amino acids 10-15, LGGQEI); SEQ ID NO: 6, (UCRP amino acids 39-44, PAFQQR); and SEQ ID NO:8, (UCRP amino acids 73-78, LVVQNC); Conformation specific domain 3: SEQ ID NO: 9 (UCRP amino acids 79-84, ISILVR);

SEQ ID NO: 13 (UCRP amino acids 95-99, VQLKQ); SEQ ID NO: 16 (UCRP amino acids 137-141 (YGLMK); Conformation specific domain 4: SEQ ID NO: 12 (UCRP amino acids 87-91, KGRSS); SEQ ID NO: 14 (UCRP amino acids 115-120, QADQFW); and SEQ ID NO: 18, 148-153 (NLRLRG). [0046] In summary, any amino acid sequence or fragment from within any region of the six predicted antigenic regions (Regions I-VI), or conformation specific domains one through four will be useful an immunogen or in any combination as immunogens for generating UCRP specific antibodies of the present invention. In particular, it may be useful to utilize certain combinations of sequences in combination as immunogens to generate the desired antibodies, such as SEQ ID NOs:l, 3, and 7; or SEQ ID NOs:2, 6, and 8; or SEQ ID NOs:9, 13, and 16; or SEQ ID NOs:12, 14, and 18. It is predicted that each one of these combinations may represent specific conformational regions, surface exposed sequences, or epitopes of UCRP that would be good targets for the generation of specific UCRP reactive antibodies of the present invention.

II. Assays for UCRP in the Detection of Pregnancy

A. Immunologic Detection of UCRP [0047] The present invention entails the use of antibodies in the immunologic detection of UCRP. Various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. 1987. Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA). Among the most useful systems are various well known immunochromatographic assays, including those known in the art as a "lateral flow assay", which can be defined as an immunochromatographic determination of the presence or absence of an antigen in a sample from an animal by combining the sample with a coloring agent-coupled antibody to the antigen, allowing the resulting combination to migrate into a first region containing a second portion of the antibody which is not coupled to a coloring agent, so that the appearance of color in the first region indicates that the antigen is present in the sample, and allowing the combination to migrate from the first region into a second region containing an antibody to the first antibody, so that the appearance of color in the second region, together with the absence of color in the first region, indicates that the antigen is not present in the sample. However, it will be readily appreciated that this invention is not limited to the use of such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used in connection with the present invention.

[0048] In general, antibody-based methods for detecting an antigen include obtaining a sample suspected of containing a protein, peptide or antibody, and contacting the sample with an antibody or protein or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of antigen-antibody complexes. [0049] Contacting the chosen biological sample with the protein, peptide or antibody under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with UCRP. After this time, the UCRP antibody mixture will be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected. [0050] In general, the detection of antigen-antibody complexes is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art. [0051] Usually, the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the UCRP-specific first antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[0052] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the UCRP antibody is used to form secondary immune complexes, as described above. The second binding ligand contains an enzyme capable of processing a substrate to a detectable product and, hence, amplifying signal over time. After washing, the secondary immune complexes are contacted with substrate, permitting detection.

B. ELISA [0053] As a part of the practice of the present invention, the principles of an enzyme-linked immunoassay (ELISA) may be used. ELISA was first introduced by Engvall and Perlmann (1971) and has become a powerful analytical tool using a variety of protocols (Engvall, 1980; Engvall, 1976; Engvall, 1977; Gripenberg et al., 1978; Makler et al, 1981; Sarngadharan et al, 1984). ELISA allows for substances to be passively adsorbed to solid supports such as plastic to enable facile handling under laboratory conditions. For a comprehensive treatise on ELISA the skilled artisan is referred to "ELISA; Theory and Practice" (Crowther, 1995 incorporated herein by reference).

[0054] The sensitivity of ELISA methods is dependent on the turnover of the enzyme used and the ease of detection of the product of the enzyme reaction. Enhancement of the sensitivity of these assay systems can be achieved by the use of fluorescent and radioactive substrates for the enzymes. Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent 4,452,901 (western blot), both incorporated herein by reference. Other assays include immuno-precipitation of labeled ligands and immuno-cytochemistry, both in vitro and in vivo. [0055] In a preferred embodiment, the invention comprises a "sandwich" ELISA, where anti- UCRP antibodies are immobilized onto a selected surface, such as a well in a polystyrene microtiter plate or a dipstick. Then, a test composition suspected of containing UCRP, e.g., a clinical sample, is contacted with the surface. After binding and washing to remove non- specifically bound proteins, the bound antigen may be detected by a secondary antibody to the UCRP.

[0056] In another exemplary ELISA, polypeptides from the sample are immobilized onto a surface and then contacted with the anti-UCRP antibodies. After binding and washing to remove non-specifically bound proteins, the bound antibody is detected. Where the initial antibodies are linked to a detectable label, the primary immune complexes may be detected directly. Alternatively, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label. [0057] Another ELISA in which the UCRP is immobilized involves the use of antibody competition in the detection. In this ELISA, labeled antibodies are added to the wells, allowed to bind to the UCRP, and detected by means of their label. The amount of UCRP in a sample is determined by mixing the sample with the labeled antibodies before or during incubation with coated wells. The presence of UCRP in the sample acts to reduce the amount of antibody available for binding to the well, and thus reduces the ultimate signal.

[0058] Regardless of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder or rabbit serum. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

[0059] In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control human cancer and/or clinical or biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0060] "Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG), evaporated or powdered milk, and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

[0061] The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about lh to 2h to 4 h, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.

[0062] To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for

2 h at room temperature in a PBS-containing solution such as PBS-Tween).

[0063] After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H O₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.

[0064] A variant of ELISA is the enzyme-linked coagulation assay, or ELCA (U.S. Patent

4,668,621, incorporated herein by reference), which uses the coagulation cascade combined with the labeling enzyme RVV-XA as a universal detection system. The advantage of this system for the current invention, is that the coagulation reactions can be performed at physiological pH in the presence of a wide variety of buffers. It is therefore possible to retain the integrity of complex analytes.

Amino Acid 3-Letter Code 1-Letter Code

Glycine Gly G

Alanine Ala A

Valine Val V

Leucine Leu L

Isoleucine He I

Proline Pro P

Cysteine Cys C

Methionine Met M

Phenylalanine Phe F

Tryptophan Trp W

Serine Ser S

Threonine Thr T

Tyrosine Tyr Y

Asparagine Asn N

Glutamine Gin Q

Lysine Lys K

Arginine Arg R

Histidine His H

Aspartic Acid Asp D

Glutamic Acid Glu E REFERENCES

[0065] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

Austin et al, Biology of Reproduction, 54:600-606, 1996.

Austin et al. Endocrine, 5:191-197, 1996.

Austin et al. Direct Sequence submission from Animal Science, University of Wyoming,

Laramie, WY 82071, USA, 1997.

Beal et al. JAnim Sci., 70(3):924-9, 1992.

Cameron and Ma mo, Austr. Vet. J., 70:109-111, 1993.

Chandrasekaran et al, Indian Veterinary Journal, 67:87-87, 1990.

Chou and Fasman, Adv. Enzym., 47:45-148, 1978.

Crowther, In: Methods in Molecule Biology, Vol. 42, Humana Press; New Jersey, 1995.

Emini etal. J. Virol, 55(3): 836-839, 1985.

Engvall and Perlmann, Immunochem., 8:871-873, 1971.

Engvall, lancet, 2(8000): 1410, 1976.

Engvall, MedBiol, 55 (4): 193 -200, 1977.

Engvall, Methods Enzymol, 70(A):419-39, 1980.

Garnier et al. J. Mol. Biol, 120:97-120, 1978.

Gήpenberg et l., ScandJ Immunol., 7(2):151-7, 1978.

Hansen etal. Endocrinology, 138:5079-5082, 1997

Hansen, et al., J. Reproduction and Fertility Supplement, 54:329-339, 1999.

Hatzidakis et al., J. Reprod. Fertil, 98:235-240, 1993.

Holdsworth et al, J. Endocrin., 95:7-12, 1982.

Hopp and Woods, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828, 1981.

Jameson, B.A. and Wolf, H. CABIOS, 4:181-186, 1988.

Johnson et al. Biol. Reproduction, 61:312-318, 1999.

Karplus and Schulz, Naturwissens-chaflen, 72: 212-213, 1985.

Marchler-Bauer et al, Nucleic Acid. Res. 31:383-387, 2003.

Marchler-Bauer et al, Nucleic Acid. Res., 30:281-283, 2002.

Margalit et al. J. Immuno 138:2213-2229, 1987.

Markusfeld et al., Br. Vet. J., 146: 504-508, 1990. Naivar et al, Biology of Reproduction, 52:848-854, 1995.

Nakamura et al, In: Handbook of Experimental Immunology (4^th Ed.), Weir et al. (Eds.),

Blackwell Scientific Publ., Oxford, 1:27, 1987. Oltenacu et al., J. Dairy Set, 73:2826-2831, 1990. Pru et al, Biology of Reproduction, 63:619-628, 2000. Rothbard, J.B., and Taylor, W.R. EMBOJ. 7: 93-100, 1988. Sarngadharan et al, Princess Takamatsu Symp., 15:301-8, 1984. Sette et al. Proc. Natl Acad. Sci USA, 86:3296-3300, 1989. Streenan and Diskin, In: Embryonic Mortality in Farm Animals, Sreenan and Diskin (Eds.),

Martinus Nijhoff Publishers, 1-11, 1986. Warnick et al, Theriogenol, 44:811-825, 1995. U.S. Patent No. 3,817,837. U.S. Patent No. 3,850,752. U.S. Patent No. 3,939,350. U.S. Patent No. 3,996,345. U.S. Patent No. 4,277,437. U.S. Patent No. 4,275,149. U.S. Patent No. 4,366,241. U.S. Patent No. 4,367,110. U.S. Patent No. 4,452,901. U.S. Patent No. 4,668,621.

U.S. Provisional Patent Application 60/435,540 filed December 19, 2002, now pending Serial No. PCT/US03/40192.

Claims

CLAIMS:

1. A method for predicting antigenic sequences of an ubiquitin cross-reactive protein (UCRP) comprising:

(a) selecting an analysis program that predicts antigenic amino acid sequences;

(b) inputting the UCRP amino acid sequence into the analysis program;

(c) obtaining the predicted antigenic amino acid sequences;

(d) aligning the UCRP sequences with protein sequences for which X-ray crystal structure data is known and that share at least 80% sequence identity with said UCRP protein to obtain predicted X-ray crystal structure data for UCRP;

(e) comparing the predicted antigenic amino acid sequences of the UCRP protein obtained in step (c) to the three dimensional X-ray crystal structure data of step (d); and

(f) identifying antigenic UCRP amino acid sequences based upon the comparative data from step (e).

2. The method of claim 1 wherein said antigenic UCRP amino acid sequences are within at least one of six antigenic regions comprising: a. SEQ ID NO:28, (UCRP amino acids 4-11); b. SEQ ID NO:29, (UCRP amino acids 19-33); c. SEQ ID NO:30, (UCRP amino acids 44-55); d. SEQ ID NO:31, (UCRP amino acids 83-97); e. SEQ ID NO:32, (UCRP amino acids 97-116); or f. SEQ ID NO:33, (UCRP amino acids 124-143).

3. The method of claim 2 wherein said antigenic UCRP amino acid sequences are selected from the group consisting of: SEQ ID NO: 1, (GDLTV, amino acids 3-7 of UCRP); SEQ ID NO: 2, (LGGQEI, amino acids 10-15 of UCRP); SEQ ID NO: 3, (LRDSM, amino acids 19-23 of UCRP); SEQ ID NO: 4, (RDSMT, amino acids 20-24 of UCRP); SEQ ID NO: 5, (KQF, amino acids 29-31 of UCRP); SEQ ID NO: 6, (PAFQQR, amino acids 39-44 of UCRP); SEQ ID NO: 7, (QGLRAG, amino acids 63-68 of UCRP); SEQ ID NO: 8, (LVVQNC, amino acids 73-78 of UCRP); SEQ ID NO: 9, (ISILVR, amino acids 79-84 of UCRP); SEQ ID NO: 10, (RNDKGR, amino acids 84-89 of UCRP); SEQ IDNO:ll, (DKGRSS, amino acids 86-91 of UCRP); SEQ ID NO: 12, (KGRSS, amino acids 87-91 of UCRP); SEQ ID NO: 13, (VQLKQ, amino acids 95-99 of UCRP); SEQ ID NO: 14, (QADQFW, amino acids 115-120 of UCRP); SEQ ID NO:15, (EEYG, amino acids 135-138 of UCRP); SEQ ID NO:16, (YGLMK, amino acids 137-141 of UCRP); SEQ ID NO:17, (MKG, amino acids 140-142 of UCRP); SEQ ID NO:18, (NLRLRG, amino acids 148-153 of UCRP); SEQ ID NO: 19, (LAH, amino acids 45-47 of UCRP); SEQ ID NO:20, (DSRE, amino acids 49-52 of UCRP); SEQ ID NO:21, (QTVA, amino acids 99-102 of UCRP); SEQ ID NO:22, (KQQ, amino acids 105-107 of UCRP); SEQ ID NO:23, (QKER, amino acids 110-113 of UCRP); SEQ ID NO:24, (MDDEH, amino acids 128-132 of UCRP); SEQ ID NO:25, (VSELK, amino acids 25-29 of UCRP); SEQ ID NO:26, (PEYVQ, amino acids 92-97 of UCRP);

SEQ ID NO:27, (EGRPMD, amino acids 124-129 of UCRP); and combinations thereof.

An antibody generated by using at least one of the antigenic UCRP amino acid sequences determined by the method of claim 1, as an immunogen. The antibody of claim 4, wherein said antigenic UCRP amino acid sequences are within at least one of six antigenic regions comprising: a. SEQ ID NO:28, (UCRP amino acids 4-11); b. SEQ ID NO:29, (UCRP amino acids 19-33); c. SEQ ID NO:30, (UCRP amino acids 44-55); d. SEQ ID NO:31, (UCRP amino acids 83-97); e. SEQ ID NO:32, (UCRP amino acids 97-116); or f. SEQ ID NO:33, (UCRP amino acids 124-143).

The antibody of claim 5, wherein said antigenic UCRP amino acid sequences are selected from the group consisting of:

SEQ ID NO: 1, (GDLTV, amino acids 3-7 of UCRP);

SEQ ID NO: 2, (LGGQEI, amino acids 10-15 of UCRP);

SEQ ID NO: 3, (LRDSM, amino acids 19-23 of UCRP);

SEQ ID NO: 4, (RDSMT, amino acids 20-24 of UCRP);

SEQ ID NO: 5, (KQF, amino acids 29-31 of UCRP);

SEQ ID NO: 6, (PAFQQR, amino acids 39-44 of UCRP);

SEQ ID NO: 7, (QGLRAG, amino acids 63-68 of UCRP);

SEQ ID NO: 8, (LVVQNC, amino acids 73-78 of UCRP);

SEQ ID NO: 9, (ISILVR, amino acids 79-84 of UCRP);

SEQ ID NO: 10, (RNDKGR, amino acids 84-89 of UCRP);

SEQ ID NO:l 1, (DKGRSS, amino acids 86-91 of UCRP);

SEQ ID NO: 12, (KGRSS, amino acids 87-91 of UCRP);

SEQ ID NO: 13, (VQLKQ, amino acids 95-99 of UCRP);

SEQ ID O:14, (QADQFW, amino acids 115-120 of UCRP);

SEQ ID NO:15, (EEYG, amino acids 135-138 of UCRP);

SEQ ID NO:16, (YGLMK, amino acids 137-141 of UCRP);

SEQ ID NO: 17, (MKG, amino acids 140-142 of UCRP);

SEQ ID NO:18, (NLRLRG, amino acids 148-153 of UCRP);

SEQ ID NO: 19, (LAH, amino acids 45-47 of UCRP); SEQ ID NO:20, (DSRE, amino acids 49-52 of UCRP); SEQ ID NO:21, (QTVA, amino acids 99-102 of UCRP); SEQ ID NO:22, (KQQ, amino acids 105-107 of UCRP); SEQ ID NO:23, (QKER, amino acids 110-113 of UCRP); SEQ ID NO:24, (MDDEH, amino acids 128-132 of UCRP); SEQ ID NO:25, (VSELK, amino acids 25-29 of UCRP); SEQ ID NO:26, (PEYVQ, amino acids 92-97 of UCRP);

SEQ ID NO:27, (EGRPMD, amino acids 124-129 of UCRP); and combinations thereof.

7. The antibody of claim 6, generated by using at least two of the amino acid sequences selected from the group consisting of SEQ ID NOs:l, 3, and 7, as immunogens.

8. The antibody of claim 6, generated by using at least two of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 6, and 8, as immunogens.

9. The antibody of claim 6, generated by using at least two of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9, 13, and 16, as immunogens.

10. The antibody of claim 6, generated by using at least two of the amino acid sequences selected from the group consisting of SEQ ID NOs: 12, 14, and 18, as immunogens.

11. The antibody of claim 6, generated by using SEQ ID NOs: 1, 3, and 7, as immunogens.

12. The antibody of claim 6, generated by using SEQ ID NOs:2, 6, and 8, as immunogens.

13. The antibody of claim 6, generated by using SEQ ID NOs: 9, 13, and 16, as immunogens.

14. The antibody of claim 6, generated by using SEQ ID NOs: 12, 14, and 18 as immunogens.

15. The antibody of any one of claims 4-14 that is a monoclonal antibody.

16. The antibody of any one of claims 4-14 that is a polyclonal antibody.

17. An antibody of claim 4 which immunoreacts with (a) SEQ ID NO: 5 and an additional L at amino acid 28 of UCRP or EL at amino acids 27-28 of UCRP and/or an additional IA at amino acids 32-33 of UCRP; (b) SEQ ID NO:15 and an additional L at amino acid 134 of UCRP and/or an additional L at amino acid 139 of UCRP; (c) SEQ ID NO: 17 and an additional C at amino acid 143 of UCRP; (d) SEQ ID NO: 19 and an additional R at amino acid 44 of UCRP; SEQ ID NO: 21 and an additional K at amino acid 98 of UCRP or LK at amino acids 97-98 of UCRP and/or an additional E at amino acid 103 of UCRP; SEQ ID NO: 22 and an additional V at amino acid 108 of UCRP; and/or SEQ ID NO: 23 and an additional V at amino acid 114 of UCRP.

18. A method for early detection of pregnancy in an ungulate animal suspected of being pregnant comprising:

(a) obtaining a sample from said animal;

(b) measuring the level of ubiquitin cross reactive protein (UCRP) in said sample with an antibody of any of claims 1 through 17; wherein elevated levels of UCRP indicate that said animal is pregnant.

19. The method of claim 18, wherein the ungulate is bovine.

20. The method of claim 19, wherein the sample comprises whole blood, plasma, saliva, urine, milk, or serum of the bovine .

21. The method of claim 19, in which the sample is obtained from the bovine from about day 15 to about day 30 after the bovine has been inseminated.

22. The method of claim 19, in which the sample is obtained from the bovine from about day 15 to about 20 after the bovine has been inseminated.

23. The method claim 19, in which the sample is serum and a level of UCRP above about 0.5 ng/ml in the serum indicates that the bovine is pregnant.

24. The method claim 19, in which the sample is serum and a level of UCRP above about 1 ng/ml in the serum indicates that the bovine is pregnant.