WO2004059282A2 - Methode et moyen de detection precoce de gestation chez des animaux a l'aide d'essais combines - Google Patents

Methode et moyen de detection precoce de gestation chez des animaux a l'aide d'essais combines Download PDF

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Publication number
WO2004059282A2
WO2004059282A2 PCT/US2003/040192 US0340192W WO2004059282A2 WO 2004059282 A2 WO2004059282 A2 WO 2004059282A2 US 0340192 W US0340192 W US 0340192W WO 2004059282 A2 WO2004059282 A2 WO 2004059282A2
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ucrp
animal
progesterone
sample
insemination
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PCT/US2003/040192
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WO2004059282A3 (fr
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Mathialagan Nagappan
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Monsanto Technology Llc
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Priority to AU2003297304A priority Critical patent/AU2003297304A1/en
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Publication of WO2004059282A3 publication Critical patent/WO2004059282A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates generally to the fields of veterinary medicine, reproductive biology and diagnostics. More specifically, the present invention relates to improved methods for early stage pregnancy detection in animals, particularly in bovines and other ruminants.
  • Pregnancy diagnosis is an important component in 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 et al, 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.
  • progesterone milk assay is the most cost effective for the producer (Oltenacu et al, 1990; Markusfeld et al, 1990). Next best is rectal palpation, performed at day 50 post-insemination (Oltenacu et al, 1990).
  • the immunoassay for PSP-B/boPAGl/PSP60 has two advantages. First, it can detect pregnancy relatively early. Second, interpretation of the assays does not require knowledge of the exact date of service, since boPAG-1 immunoreactive molecules are always present in the maternal serum of pregnant cows by day 28, and concentrations increase as pregnancy advances (Sasser et al, 1986; Mialon et al, 1993; Mialon et al, 1994).
  • the test can be carried out in dairy cows at day 30 only if artificial insemination ("Al") is performed at 45-70 days post-partum.
  • Al artificial insemination
  • Analysis of other BoPAGs in particular has exhibited potential for use in pregnancy testing.
  • Such tests can yield high false positive rates. This error rate occurs because the PAG test is done at day 25 of pregnancy. However, some embryos die between day 20 and 30 of pregnancy. This dying tissue can probably produce some PAG. Thus, the cow is PAG positive, but the embryo is dead. The results of this can be a false positive rate that may be unacceptable within some commercial breeding programs. There is, therefore, a need for pregnancy tests with improved accuracy.
  • bovine ubiquitin cross-reactive protein also known as bovine IFN-stimulated gene product- 17, ISG17
  • bovine IFN-stimulated gene product- 17, ISG17 bovine IFN-stimulated gene product- 17, ISG17
  • IFN-tau secreted by the trophoblast layer of the conceptus, functions as a maternal signal denoting recognition of pregnancy in ruminants.
  • IFN-alpha and IFN-beta Type I IFN class of proteins
  • 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.
  • IFN-stimulated gene product ISG15
  • UCRP ubiquitin cross-reactive protein
  • Bovine ISG17 (or 1 bUCRP) is a bovine ortholog of human ISG15. Sequence comparison showed that bUCRP has 60% sequence identity with human ISG15 (see, Hansen et al, 1997).
  • ISG17 may be released into peripheral circulation via the histotroph (also known as uterine milk) which nourishes the embryo until the placenta forms.
  • histotroph also known as uterine milk
  • the invention provides methods for the early detection of pregnancy in livestock such as ungulates (e.g., hoofed animals), and specifically in ruminants such as cows, sheep and goats.
  • methods are provided for the early detection of pregnancy in a bovine animal comprising: (a) obtaining a sample from the bovine animal; (b) measuring the level of ubiquitin cross-reactive protein (UCRP), the bovine form of which is also known as interferon-stimulated gene product-17 (ISG17); or (c) measuring the level of UCRP and measuring the level of progesterone in the sample, wherein elevated levels ISG17 and progesterone indicate that the bovine animal is pregnant.
  • UCRP ubiquitin cross-reactive protein
  • ISG17 interferon-stimulated gene product-17
  • the sample may be from any biological material, including saliva, serum, blood, milk or urine.
  • the sample may be obtained from the animal at days 16 to 28 post-insemination, including about day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, post-insemination:
  • the UCRP that is analyzed is present at increased early pregnancy (i.e. between about day 16 and about day 28) and then decreases to constitutive levels shortly thereafter, returning to pre-pregnancy levels by approximately day 28 and afterward.
  • the measuring may comprise immunologic detection, including detecting a UCRP with polyclonal antisera.
  • the polyclonal antisera is prepared against immuno-reactive fragments of a UCRP, such as ISG17, or comprises detecting a UCRP with a monoclonal antibody preparation.
  • Immunologic detection may be carried out using any technique, including ELISA, RIA, and Western blot.
  • the ELISA may comprise a sandwich ELISA comprising binding of a UCRP 11916.0056.00PC00
  • the enzyme is alkaline phosphatase or horseradish peroxidase.
  • an elevated level of UCRP that is detected is selected as a cut-off for determination that the animal is pregnant is selected so as to provide a minimal number of false-positive and/or false-negative results while simultaneously providing early detection in accordance with the present invention.
  • the cut-off level of UCRP selected for determination that an animal is pregnant is preferably from at least about 0.5 ng/ml to about 30 ng/ml. Typically, the value is not higher than 20 ng/ml and more preferably it is the level is not higher than 10 ng/ml.
  • the serum UCRP cut-off level is between about 1.0 and 5.0 ng/ml. In the most preferred embodiment the cut-off value is 2.0 ng/ml or more.
  • measuring UCRP levels may comprise, for example, nucleic acid hybridization, including Northern blotting and nucleic acid hybridization comprises amplification.
  • the amplification may comprise RT-PCR.
  • measuring progesterone levels may also comprise immunologic detection.
  • immunologic detection may comprise detecting progesterone with polyclonal antisera or detecting progesterone with a monoclonal antibody preparation.
  • Immunologic detection may be carried out using any technique, including ELISA, RIA, and Western blot.
  • the ELISA may comprise a sandwich ELISA comprising binding of a progesterone to a first antibody preparation fixed to a substrate and a second antibody preparation labeled with an enzyme.
  • the enzyme is alkaline phosphatase or horseradish peroxidase.
  • the level of UCRP considered to be "elevated" in pregnant animals as compared with non-pregnant animals may range from about 0.5 to about 20 ng/ml of serum, including from about 2 ng/ml to about 10 ng/ml. However, typically basal levels in non-pregnant animals range from 0.5 to 2 ng/ml, whereas the level in pregnant animals is from 2 to 20 ng/ml.
  • the elevated level of progesterone that is detected may, in certain embodiments of the invention, comprise about 2-5 ng/ml of serum. In particularly preferred aspects of this embodiment the elevated level of progesterone is about 3 ng/ml of serum. 11916.0056.00PC00
  • a sample is obtained from a bovine animal between about day 16 and about day 28 post-insemination, and the elevated levels of UCRP are those greater than about 2 ng/ml of serum and elevated levels of progesterone include those of about 2-5 ng/ml of serum (preferably about 3 ng/ml of serum) or greater.
  • the sample is obtained between about day 18 and about day 23 after insemination.
  • the sample is obtained between about day 18 and about day 20.
  • a positive control sample may also be obtained from a pregnant bovine animal, as may a negative control sample from a non-pregnant bovine animal.
  • the method may further comprise measuring UCRP and progesterone levels from a second sample from the bovine animal at a second point in time.
  • the invention provides a method of making a breeding decision for a bovine animal comprising: (a) obtaining a sample from the bovine animal, wherein the bovine animal is suspected of being pregnant; (b) measuring the level of ISG17 in the sample; and (c) measuring the level of progesterone in the sample, wherein: (i) elevated levels of ISG17 and progesterone indicate that the bovine animal is pregnant, and no further steps need be taken; (ii) non-elevated levels of ISG17 and progesterone indicate that the bovine animal is not pregnant, and should be injected with gonadotropin-releasing hormone (GnRH), and about seven days later, injected with prostaglandin F 2 ⁇ (PGF), followed by re-insemination; (iii) elevated levels of ISG17 and non-elevated levels of progesterone indicate that the bovine animal is not pregnant due to early embryo death and should be injected with GnRH, and about seven days later, injected with PGF, followed by
  • the method may also further comprise steps (ii), (iii) and (iv), about 48 hours after PGF injection and before re-insemination, administering a second injection of GnRH.
  • the method may also further comprise, prior to step (a), inseminating the bovine animal.
  • Various embodiments contemplate methods for the diagnosis of pregnancy in ruminants and methods for making breeding decisions in ruminants which comprise the detection of the levels of a UCRP (such as ISG17), in a biological sample, either by itself, or in combination with a second antigen (the second antigen may be progesterone or some other pregnancy-specific antigen). 11916.0056.00PC00
  • Figure 1 shows the typical results of tests of the present invention carried out on both pregnant and open cows.
  • the invention overcomes the limitations of the prior art by providing a reliable test for early pregnancy diagnosis and methods for use thereof.
  • a reliable yet simple pregnancy test for cattle has long been sought.
  • Typical prior test 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 thus fallen short of expectations in terms of their practical, on-farm use.
  • the animals remain "open” for two or three estrous cycles. It present invention minimizes this "open” time by allowing for the determination of pregnancy, or non-pregnancy, as early as the first estrus following a breeding attempt.
  • ubiquitin cross-reactive protein (UCRP) (the bovine version of which is also known of as interferon-stimulated gene product- 17, or ISG17, the nucleotide sequence of the mRNA encoding the bovine ISG17 protein, as well as the sequence of the protein is provided infra in the section entitled Sequence Listing) in a biological sample of an animal suspected of being pregnant.
  • the UCRP may be analyzed by itself to determine the pregnancy status of the animal suspected of being pregnant.
  • the UCRP levels are analyzed immunologically by detecting the UCRP levels with an antibody which was raised to full-length UCRP (either native or recombinant protein).
  • the antibody specifically recognizes full length UCRP protein. 11916.0056.00PC00
  • the UCRP levels present in the biological samples may be determined in conjunction with the determination of the levels of one, or more other pregnancy specific compounds.
  • the levels of UCRP are determined in conjunction with a determination of the progesterone levels in a biological sample from the same animal in order to provide for the early and accurate diagnoses of pregnancy in bovine and other ruminants.
  • the inventor has found that by assaying for both progesterone and UCRP, early pregnancy diagnosis is possible with a high degree of accuracy. This is because the combined test measures a conceptus-stimulated component, UCRP, and a maternal component, progesterone, both of which are elevated upon the establishment of a successful pregnancy in cattle ruminant species.
  • pregnancy diagnosis is an important component in reproductive management of livestock, particularly in the dairy industry where a high proportion of artificial inseminations fail and additional days open reduce the net operating income to the producer.
  • kits for the diagnosis of pregnancy or non-pregnancy in an animal suspected to be in the early stages of pregnancy.
  • the subject animal is a ruminant selected from the group consisting of bovine, ovine, and caprine.
  • the animal is bovine.
  • the animal is a dairy heifer or dairy cow.
  • the diagnosis of pregnancy or non- pregnancy is made by analyzing a biological sample from the animal suspected of being in the early stages of pregnancy. The analysis is done by detecting the presence and/or amount of ubiquitin cross-reactive protein (UCRP) and progesterone in the biological sample from animals, including bovines, suspected of being in the early stages pregnancy.
  • the biological sample comprises serum, plasma, blood, saliva, urine, milk, or any other suitable sample from the subject animal which is compatible with the present invention.
  • the biological sample is from blood, serum, or plasma.
  • the biological sample is obtained from the animal suspected of being pregnant from 15 to 28 days after natural breeding or artificial 11916.0056.00PC00
  • the sample may be collected at any of days 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 after breeding. More preferably the sample may be collected at about days 16 to 23 after breeding, and even more preferably 18-21 days after breeding.
  • the animal whose pregnancy status is to be determined is a ruminant. More preferably the animal is a bovine, ovine, or caprine animal. Even more preferably the animal is bovine and most preferably the animal is a beef or dairy cow.
  • the pregnancy test is carried out by detecting UCRP and/or progesterone by immunological methods.
  • the antibodies are raised against either full-length native or full-length recombinant UCRP.
  • the full length UCRP is full length ISG17.
  • the immunogen is full-length bovine ISG17 and the antibodies are monoclonal antibodies.
  • UCRP in combination with progesterone can be used advantageously expressed in early stages of pregnancy and, therefore, can be used as markers in the detection of pregnancy at an early stage.
  • UCRP may be used individually or in combination with other diagnostic methods to provide a diagnostic evaluation of pregnancy.
  • the UCRP, bISG17 may be used. It is envisioned that UCRPs from other species, will also prove useful, alone or in combination, for similar purposes.
  • ISG17 As noted above, the presence of detectable ISG17 above background levels in the maternal circulation indicates the presence of a conceptus in the uterus. However, the circulating levels of ISG17 will diminish when the conceptus ceases to produce IFN-tau, which typically 11916.0056.00PC00
  • the conceptus begins to produce IFN-tau from about days 13 to 21 of pregnancy, with production peaking at around days 16 to 18.
  • the raise in serum ISG17 levels then follows in response to the secreted IFN-tau. A raise in ISG17 can be observed beginning at about day 15 of pregnancy, with levels peaking at about days 18 to 20. Thereafter, the ISG17 level begins to decline.
  • the serum progesterone concentration in the pregnant cows is usually about 3 ng/ml or above on day 16 and continues to rise, thereafter. If a cow is not pregnant, the serum progesterone concentration decline significantly from day 16 onwards and drops to below 3 ng/ml (in most cases below 1.0 ng/ml) by day 19 to day 23 (Santos et al, 2002; Ayalon, 1978; Weibold, 1988).
  • One embodiment of the present invention is directed to a method comprising a combined testing for ISG17 and progesterone used to determine the pregnancy status of a cow.
  • a background threshold level e.g. >2 ng/ml
  • serum progesterone value of from about 2-5 ng/ml or above
  • the cow is likely to indicate that a cow is pregnant.
  • the serum progesterone level is about 3 ng/ml or higher.
  • ISG17 is absent in the serum or present at a level below the "threshold level” and the animal. has a serum progesterone values below about 2-5 ng/ml, and preferably below about 3 ng/ml, the animal is considered to be not pregnant or "open.”
  • the specificity and sensitivity of the combined test is likely to range from 75 to 95% when testing is done from day 16 to day 23 following artificial insemination. 11916.0056.00PC00
  • the specificity and sensitivity of the combine test would be 70 to 90% due to a high incidence of early embryonic loss compared to day 45 pregnancy status (palpation data).
  • Various embodiments of the present invention comprise methods for detecting the levels of UCRP and/or progesterone that are well known and widely used in the art. Particular aspects of this embodiment include determination of UCRP and/or progesterone levels using immunological and/or nucleic acid based methods.
  • Contemplated embodiments of the present invention include those employing the use of antibodies in the immunologic detection of UCRP and/or progesterone.
  • Various useful immunodetection methods have been described in the scientific literature, such as, e.g., Nakamura et al. (1987; incorporated herein by reference).
  • Immunoassays in their most simple and direct sense, are binding assays.
  • Certain preferred embodiments include the use of immunoassays including various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA).
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections also is contemplated as useful for the present invention. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used in connection with the present invention.
  • immunobinding methods 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 immunocomplexes.
  • Preferred samples, according to the present invention are fluids, such as milk, urine, blood, serum or saliva.
  • the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the UCRP or progesterone-specific first antibody.
  • 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.
  • 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 or progesterone 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.
  • Progesterone can also be detected in accordance with the invention using various commercially available detection kits.
  • the COAT-A-COUNTTM progesterone kit used by the inventors, which is available from Diagnostics Products Corporation (Los Angeles, CA).
  • Examples of other assays that have been described include the immunoenzymatic technique described, for example, by Stefanakis et al., (1994) and by Stanley et al. (1986); and salivary progesterone level assays described, for example, by Lu et al, (1997) and Vienravi et al., 1994. 11916.0056.00PC00
  • the UCRP detected is bovine ISG17 and the WSG17 and the progesterone are detected using monoclonal antibodies to either bISG17 or progesterone as part of an ELISA.
  • ELISA enzyme-linked immunoassay
  • ELISA allows for substances to be passively adsorbed to solid supports such as plastic to enable facile handling under laboratory conditions.
  • ELISA ELISA; Theory and Practice
  • 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). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
  • the invention comprises a "sandwich" ELISA, where anti-UCRP antibodies, preferably anti-bISG17 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, such as blood or serum, is contacted with the surface. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen may be detected by a second antibody to the UCRP.
  • a test composition suspected of containing UCRP e.g., a clinical sample, such as blood or serum
  • 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 immune complexes, the bound antibody is detected.
  • the primary immune complexes may be detected directly.
  • the immune complexes may be detected using a second 11916.0056.00PC00
  • the second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the UCRP is immobilized involves the use of antibody competition in the detection.
  • 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.
  • 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.
  • 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.
  • BSA bovine serum albumin
  • 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.
  • a secondary or tertiary detection means rather than a direct procedure.
  • 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.
  • 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)/TWEENTM. These added agents also tend to assist in the reduction of nonspecific background. 11916.0056.00PC00
  • 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 1 h to 2 h to 4 h, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at approximately 4°C.
  • 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.
  • 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 O 2 , 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.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H O 2 , 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.
  • a variant of ELISA is the enzyme-linked coagulation assay, or ELCA (U.S. Patent
  • nucleic acids which encode UCRPs, such as MSG17, and/or proteins involved in the biosynthesis of progesterone to determine the levels of the corresponding proteins.
  • methods include Northern assays and RT-PCR. The following describe methods relevant to the detection and quantification of such nucleic acids.
  • Hybridization The use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and
  • nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and or RNAs or to provide primers for amplification of DNA or RNA from samples.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts.
  • conditions can be rendered more stringent by the addition of increasing amounts of formamide. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • Hybridization conditions can be readily manipulated depending on the desired results. In other embodiments, hybridization may be achieved under conditions of, for example,
  • nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for 11916.0056.00PC00
  • indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.
  • the probes or primers described herein will be useful as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art.
  • analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA. 11916.0056.00PC00
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.
  • Pairs of primers designed to selectively hybridize to nucleic acids corresponding to a UCRP are contacted with the template nucleic acid under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
  • hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
  • PCRTM polymerase chain reaction
  • a reverse-transcriptase coupled PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al, 1989).
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent 5,882,864. 11916.0056.00PC00
  • LCR ligase chain reaction
  • PCRTM and oligonucleotide ligase assay (OLA), disclosed in U.S. Patent 5,912,148, may also be used.
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • SDA Strand Displacement Amplification
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety.
  • European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence.
  • This scheme is not cyclic, i.e., new templates are not produced from the 11916.0056.00PC00
  • RNA transcripts resultant RNA transcripts.
  • Other amplification methods include “race” and “one-sided PCR” (Frohman, 1990; Ohara etal, 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid. Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatography There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al, 1989).
  • U.S. Patent 5,279,721, incorporated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention. 11916.0056.00PC00
  • kits This generally will comprise a probe or primers designed to hybridize specifically to individual nucleic acids and/or antibodies capable of specifically recognizing the molecules of interest in the practice of the present invention. Also included may be enzymes suitable for detecting the interaction of the antibodies with the target antigens and/or enzymes for amplifying nucleic acids, including various polymerases (reverse transcriptase, Taq, etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • polymerases reverse transcriptase, Taq, etc.
  • kits may also include enzymes and other reagents suitable for detection of specific proteins/compounds or nucleic acids or amplification products and/or for detecting antbody/ligand interaction.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or enzyme as well as for each probe, primer pair, and/or antibody.
  • One advance of the current invention is that it allows early detection of pregnancy with a low incidence of false positive results. Early detection of pregnancy is important because it allows re-breeding of animals found to not be pregnant, thereby minimizing the period during which the animal is "open.” A low incidence of false positives is necessary to allow implementation of an effective re-breeding protocol. Prior pregnancy tests typically either were unable effectively detect early pregnancy or exhibited high incidence of false positives.
  • a type of early pregnancy test which has been used is the detection of pregnancy associated antigens (PAGs).
  • PAGs pregnancy associated antigens
  • An advantage of this test is that it can be done at day 25 of pregnancy. However, some embryos die between day 20 and 30 of pregnancy and, in some cases, the dying tissue may produce PAG. Thus cows may be PAG positive but the embryo is dead. 11916.0056.00PC00
  • the inventors have found that by analyzing progesterone levels in addition to UCRP, a very low incidence ( ⁇ 5%) of false positives can be obtained. This is because the corpus luteum regresses shortly after embryo death. Thus, a cow with a dying embryo would have ISG17 but low progesterone. Because the progesterone is an absolute requirement for establishing pregnancy, a cow with low serum progesterone cannot maintain pregnancy.
  • the subject animals are ruminants, including bovine, ovine, and caprine. More preferably, the animals are beef or dairy cattle.
  • the method comprises obtaining a biological sample from an animal suspected of being pregnant.
  • the biological sample comprises a blood, serum, plasma, milk, urine, or saliva sample, but any other biological samples suitable for use with the present invention are also contemplated.
  • the sample is preferably collected 16-28 days after natural breeding or artificial insemination (collectively, "breeding") has taken place. More preferably the sample is serum collected 16-23 days after breeding and most preferably the sample is collected 18-21 days after breeding.
  • the sample is analyzed to determine the levels of UCRP (preferably bISG17 for cattle) and progesterone present in the sample. If the level of UCRP and progesterone are both elevated, as compared with non-pregnant controls, the animal is determined to be pregnant and no further action need be taken. If the levels of neither UCRP nor progesterone are elevated, this indicates that the animal is not pregnant and additional actions are required.
  • the animal should be injected with gonadotropin-releasing hormone (GnRH), and about seven days later, injected with prostaglandin F ⁇ (PGF), followed by re-insemination.
  • GnRH gonadotropin-releasing hormone
  • PEF prostaglandin F ⁇
  • the animal should be injected with GnRH, then about seven days later, injected with PGF, followed by re-insemination.
  • the method further comprises administering a (second) dose of GnRH about 48 hours after the PGF injection and before re-insemination.
  • the PGF injection is administered at day 20 post-insemination and re-insemination is carried out at day 28 post- insemination.
  • Dairy cows come into estrus once every 21 days. Cows display characteristic behaviors during estrus. Dairy workers can identify cows in estrus by these characteristic behaviors. Cows ovulate an egg about 28 hours after the onset of estrus. Most dairy cows are inseminated artificially about 12 hours after the onset of estrus so that sperm are in the reproductive tract when the cow ovulates.
  • Lactating dairy cows are watched for estrus. They are inseminated when they come into estrus so that they can become pregnant and have another calf. The efficiency with which cows are detected in estrus is low. Only about 50% of cows in estrus are actually detected by farmers. Of the cows detected in estrus and inseminated, only about 30% will become pregnant. Thus, only about 15% (50% x 30%) of ovulations result in a pregnancy. Thus, dairy reproduction can be inefficient because cows in estrus are not always seen and those inseminated do not always get pregnant. Although most cows could theoretically be artificially inseminated once every 21 days, the true insemination interval on farms is typically once every 40 to 60 days.
  • a corpus luteum (CL) is formed on the ovary and the CL secretes the hormone progesterone.
  • Progesterone can be detected in the blood of the mother is needed to maintain the pregnancy. If the egg is fertilized and the embryo grows and survives then the corpus luteum will be maintained until the end of gestation (280 days). If the egg is not fertilized or the embryo dies then the corpus luteum will regress and the cow will reenter estrus.
  • IFN-tau interferon-tau
  • IFN-tau stimulates the uterine endometrium to produce UCRP, which can be detected in the blood of the mother at about 15 to 28 days of pregnancy in bovine and other ruminants.
  • the UCRP pregnancy test is designed to detect UCRP in the blood/serum of the mother.
  • a pregnant cow will also have high progesterone in blood because she will have a corpus luteum.
  • pregnant cows will have UCRP and progesterone in their blood/serum.
  • Pregnancy testing in dairy cows has usually been done by rectal palpation (manually feeling for an embryo in the uterus).
  • the manual test is typically done 35 to 60 days after breeding. On large dairies, a veterinarian is often employed 100% time to do manual pregnancy testing.
  • the only alternative to manual testing is ultrasound testing. While, ultrasound testing can be done at 28 days after breeding, it is not routine because the equipment is expensive and testing takes longer than rectal palpation. 11916.0056.00PC00
  • Dairy cows can be injected with prostaglandin F 2 ⁇ (PGF) to regress the corpus luteum and cause estrus.
  • PGF prostaglandin F 2 ⁇
  • Cows that do not have a corpus luteum do not respond to PGF.
  • dairy cows without a corpus luteum can be injected with gonadotropin-releasing hormone (GnRH) to cause ovulation and the formation of a corpus luteum.
  • GnRH gonadotropin-releasing hormone
  • One typical method for managing dairy cows without a corpus luteum is to inject GnRH, wait 7 days (allows CL to form), inject PGF and await the cow's next estrus.
  • the assays to detect the levels of progesterone may be carried out either on biological samples collected on the same day or on biological samples collected on different days, as is convenient and/or most efficacious.
  • the assays to determine the levels of UCRP and progesterone levels are determined in a sample or samples which are collected on the same day.
  • the assays are ELISA or another antibody based assay wherein the antibodies function separately.
  • they may be located at separate positions on the assay device or the assay may utilize one separate device for UCRP and another for progesterone.
  • the method for determination of the UCRP and progesterone levels in the biological samples comprises the use of one or more "Lateral Flow Assay" device(s) wherein the two antigens are detected separately.
  • a "Lateral Flow Assay” means an immunochromatographic determination of the presence or absence of an antigen in a biological sample from an animal by: a) combining the sample with a coloring agent-coupled antibody, specific for the antigen; b) allowing the resulting combination to migrate into a first region containing a second antibody to the antigen, 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 c) 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, serves as a control which indicates that the antibody to the antigen is present, but the antigen is not present.
  • Placenta is the hallmark of the eutherian mammal. Rather than being the most anatomically conserved mammalian organ, however, it arguably is the most diverse (Haig, 1993). Placentation ranges from the invasive hemochorial type, as in the human, where the 11916.0056.00PC00
  • trophoblast surface is in direct contact with maternal blood, to the epitheliochorial (e.g., pig), where the uterine epithelium is not eroded (Amoroso, 1952).
  • epitheliochorial e.g., pig
  • the polypeptide hormones the placenta produces also vary between species (Haig, 1993; Roberts et al, 1996).
  • no group of mammals other than higher primates possesses a chorionic gonadotrophin homologous to hCG for luteal support in early pregnancy, and only the ruminant ungulates are known to produce Type I interferon as an antiluteolytic hormone (Roberts et al, 1996).
  • the endometrial luminal and glandular epithelial cells secrete several proteins including UCRP into the uterine lumen (reviewed in 'Biology of progesterone action during pregnancy recognition and maintenance of pregnancy' Spencer TE and Bazer FW., Frontiers in Bioscience 7:1879-1898, 2002).
  • Uterine flushings or endometrial tissue collected from days 16 to day 25 pregnant animals are used for purification of IFN-tau induced proteins (Hansen et al., Endocrinology 138:5079- 5082, 1997).
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • the te ⁇ n "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally- obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur. 11916.0056.00PC00
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%), about 90%), about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "- fold purification number" (i.e., 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, etc.).
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than 11916.0056.00PC00
  • Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of min, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography, or molecular sieve chromatography is a special type of partition chromatography that is based on molecular size.
  • gel chromatography The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related to molecular weight.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the " column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.). 11916.0056.00PC00
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N- acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the present invention provides for the use of a UCRP (such as bISG17) or peptides fragments thereof as antigens for the generation of polyclonal antisera and monoclonal antibodies for use in the detection of UCRP in a biological sample as part of a test for the diagnosis of pregnancy. It is envisioned that some a UCRP, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced.
  • a UCRP such as bISG17
  • peptides fragments thereof as antigens for the generation of polyclonal antisera and monoclonal antibodies for use in the detection of UCRP in a biological sample as part of a test for the diagnosis of pregnancy. It is envisioned that some a UCRP, or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more
  • compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable.
  • Preferred agents are the carriers such as keyhole limpet hemocyannin (KLH) or glutathione-S-transferase. 11916.0056.00PC00
  • Aqueous compositions of the present invention comprise an effective amount of an UCRP (such as WSG17) antigen to the host animal, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • UCRP such as WSG17
  • Such compositions may be referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the UCRP may be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and 11916.0056.00PC00
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the PAGs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl 11916.0056.00PC00
  • antibodies, specific for UCRP, or a fragment thereof may be raised against UCRP from any ruminant animal.
  • the UCRP is from a bovine, ovine, or caprine animal.
  • the UCRP is bovine ISG17 or a fragment thereof.
  • the bovine ISG17 used to as the antigen elicit the antibodies is recombinant ISG17 obtained using a published DNA sequence for ISG17 (such as the one provided in the Sequence Listing of this disclosure) and recombinant DNA techniques which are well known to those of ordinary skill in the art.
  • Such an antibody preparation can be a polyclonal or a monoclonal antibody composition, both of which are contemplated for use with preferred embodiments of the present invention.
  • Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988). While it is possible to generate a polyclonal antibody preparation which simultaneously comprises some antibodies recognizing UCRP and some recognizing progesterone. The preferred embodiment of the present invention is to generate antibodies to UCRP and progesterone separately.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a peptide or polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a peptide or polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera. 11916.0056.00PC00
  • an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific for UCRR-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular UCRP of different species may be utilized in other useful applications.
  • both polyclonal and monoclonal antibodies against UCRP or progesterone may be used in a variety of embodiments.
  • they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding antibodies to UCRP and/or proteins involved in the biosynthesis of progesterone. They may also be used in inhibition studies to analyze the effects of UCRP related peptides or progesterone related compounds in cells or animals.
  • Anti-UCRP or antibodies to progesterone pathway enzymes will also be useful in immunolocalization studies to analyze the distribution of UCRP or enzymes that participate in progesterone biosynthesis or metabolism during various cellular events, for example, to determine the cellular or tissue-specific distribution of UCRP or progesterone biosynthesis or metabolism under different points in the cell cycle.
  • a particularly useful application of such antibodies is in purifying native or recombinant UCRP, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure. 11916.0056.00PC00
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, w-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis- biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified UCRP or progesterone.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. 11916.0056.00PC00
  • mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the MAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmoblast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986). 11916.0056.00PC00
  • Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10 "6 to 1 x 10 "8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like. >
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily 11916.0056.00PC00
  • An immunoassay which is developed using antibodies raised against full-length, native or recombinant UCRP offers more specificity than one using antibodies raised to peptide fragments of UCRP. This is because antibodies raised to whole protein antigen are more likely to be conformation-specific antibodies and offer more specificity to immunoassay. In contrast, antibodies raised against peptide fragment antigens are more likely to be sequence specific rather than conformation specific and will, consequently, be more likely to cross-react with related and/or unrelated proteins thereby diminishing the immunoassay' s specificity and/or sensitivity.
  • An immunoassay developed with full-length, native or recombinant bovine ISG17 will detect WSG17 protein present in a biological sample such as serum, plasma, saliva, urine, or blood with high specificity and sensitivity.
  • a bISG17 specific immunoassay may be developed by first raising an antibody to either purified native ISG17, such as WSG17, isolated from endometrial secretions.
  • purified recombinant ISG17 produced in yeast or bacteria may be used as the antigen.
  • Recombinant protein produced in yeast has been shown to have proper folding by a bioassay (Pru et al., 2000). However, production of WSG17 in bacteria may require re-folding and the extent of re-folding could be tested with a bioassay described by Pru et al (2000).
  • Example 3 Polyclonal UCRP assay development The following section describes the means for a polyclonal antibody based assay development for UCRP.
  • the assay may be standardized using purified recombinant bISG17 as 11916.0056.00PC00
  • This assay may be used to determine the feasibility of detecting UCRP during early pregnancy using various polyclonal antibodies.
  • Antigen proteins may be prepared and isolated by any means known to those of ordinary skill in the art. Antibodies may be generated in rabbits according to the standard protocol-using purified
  • UCRP UCRP in Freund's complete adjuvant. After a two-week interval these rabbits may be boosted with the antigen with incomplete adjuvant. The rabbits can then be boosted every two weeks until sufficient antisera were collected and stored at -70 °C.
  • Polyclonal antibodies can then be affinity purified using protein A chromatography and dialyzed in PBS. Purified antibodies are then aliquoted and stored at -70 °C.
  • Example 3 Design of a combination UCRP/progesterone early pregnancy test and Results expected.
  • UCRP pregnancy induced protein
  • progesterone maternal component
  • a series of designated UCRP and progesterone cutoff levels for pregnancy determination based on given concentrations of UCRP and progesterone can be formulated. For each day post insemination as serious of analyses may be done to determine the appropriate cut off levels for serum (or other sample) levels of UCRP in cows.
  • a studies may be done which determine the serum levels of UCRP and progesterone in a statistically significant number of cows on the days following artificial insemination or sham-insemination as control.
  • a standard may be statistically calculated which provides a base line level of UCRP and progesterone, below which a cow should be considered non-pregnant and above which a cow should be considered pregnant.
  • the same data may also be used to determine the optimal window to allow for the early detection of pregnancy which provides both acceptable sensitivity and accuracy and diagnosis early enough to allow re-breeding so as to minimize the time the cow is open.
  • cutoff ranges 3 ng/ml and 2 ng/ml have been determined for progesterone.
  • the cutoff ranges were selected based on: a) pregnancy history of the cows, b) Progesterone levels during estrus cycle and pregnancy in cows.
  • Example 4 Diagnosis of Pregnancy Non-pregnancy in a bovine suspected of being pregnant.
  • Antibodies may be raised respectively against bISG17 and progesterone. This antibodies may then be employed to prepare a kit comprising, inter alia, the components necessary to provide sandwich-type ELISA assays for individually detecting the presence bISG17 and progesterone in a biological sample.
  • the pregnancy status of a bovine may then be determined by collecting a biological sample, such as serum from the cow 20 days after natural breeding or artificial insemination has taken.
  • the biological sample can then be analyzed to determine the levels of bISG17 and progesterone in the sample. Threshold levels can be individually established which will be determined to be a "positive" result (a positive result meaning that the animal is pregnant) for bISG17 and progesterone ELISA assays.
  • Threshold levels can be individually established which will be determined to be a "positive" result (a positive result meaning that the animal is pregnant) for bISG17 and progesterone ELISA assays.
  • a suitable cutoff level for designation as a "positive result" for bISG17 is 2 ng/ml or more and for progesterone the level is 3 ng/ml or more.
  • the results of the MSG17 and progesterone assays can be determined and a diagnosis of pregnant or "open” can be made in
  • Example 5 Resynchronization of Dairy Cows and Heifers after UCRP/Progesterone Pregnancy Diagnosis
  • the following a method for re-breeding cows and heifers that are diagnosed as not pregnant after a bISG17/progesterone test Generally, cattle are tested for UCRP/progesterone 16 to 28 days after natural breeding or artificial insemination and are diagnosed pregnant or non-pregnant.
  • the resynchronization method is implemented on non-pregnant cows 0 to 2 days after the ISG17/progesterone test.
  • Animals are treated in the following sequence: (i) inject prostaglandin F 2 ⁇ (PGF 2 ⁇ ; a hormone causing regression of the corpus luteum); (ii) wait two days, inject gonadotropin releasing hormone (GnRH; a hormone causing ovulation); (iii) wait 0 to 8 hours, (iv) inseminate artificially.
  • PPF 2 ⁇ prostaglandin F 2 ⁇
  • GnRH gonadotropin releasing hormone
  • Dairy cows and heifers are tested for ISG17 16 to 28 days after first artificial insemination.
  • Cattle diagnosed not pregnant are treated with 5 niL Lutalyse (25 mg PGF 2 ⁇ ), two days later were treated with 2 mL Cystorelin (100 ⁇ g GnRH), and are inseminated 0 to 8 hours after GnRH.
  • the resynchronization treatment is administered 0 to 2 days after the ISG17 test (16 to 30 days after first insemination). Pregnancy is determined 30 to 60 days after insemination.
  • Austin et al Endocrine, 5:191-197, 1996. Austin et al, Biology of Reproduction, 54:600-606, 1996.
  • Roberts et al Biol. Reprod, 54:294-302, 1996. Roberts et al. , Prog. Nucl Acid Res. Mol. Biol, 56:287-326, 1996.
  • REFERENCE 1 (residues 1 to 154) AUTHORS Austin,K.J., Pru,J.K. and Hansen,T.R.
  • AUTHORS Austin,K.J., Pru,J.K. and Hansen,T.R. TITLE Complementary deoxyribonucleic acid sequence encoding bovine ubiquitin cross-reactive protein: a comparison with ubiquitin and a 15 -kDa ubiquitin homolog JOURNAL Endocrine 5, 191-197 (1996) REMARK SEQUENCE FROM N.A.
  • TISSUE Endometrium REFERENCE 2 (residues 1 to 154) AUTHORS Perry,D.J., Austin,K.J. and Hansen,T.R. TITLE Cloning of interferon-stimulated gene 17: the promoter and nuclear proteins that regulate transcription JOURNAL Mol. Endocrinol.
  • REFERENCE 5 (residues 1 to 154) AUTHORS Hansen,T.R., Austin,K.J. and Johnson,G.A. TITLE Transient ubiquitin cross-reactive protein gene expression in the bovine endometrium JOURNAL Endocrinology 138 (11), 5079-5082 (1997) MEDLINE 98006468 PUBMED 9348245 REMARK EXPRESSION.
  • PROTEINS INVOLVED WITH RELEASE OF PROSTAGLANDIN F2-ALPHA PPF
  • PPF PROTEINS INVOLVED WITH RELEASE OF PROSTAGLANDIN F2-ALPHA
  • PPF PROTEINS INVOLVED WITH RELEASE OF PROSTAGLANDIN F2-ALPHA
  • PPF PROTEINS INVOLVED WITH RELEASE OF PROSTAGLANDIN F2-ALPHA
  • PPF PROTEINS INVOLVED WITH RELEASE OF PROSTAGLANDIN F2-ALPHA
  • PPF PROSTAGLANDIN F2-ALPHA
  • THUS PREVENT LYSIS OF THE CORPUS LUTEUM AND MAINTAIN THE PREGNANCY AS IT IS ALSO DETECTED IN UTERINE FLUSHINGS, MAY ALSO HAVE AN EXOCRINE FUNCTION. MAY ALSO, BY INDUCING THE SECRETION OF IFN- GAMMA FROM T-CELLS, AUGMENT NATURAL KILLER (NK) CELL
  • INTERFERON TAU BY THE CONCEPTUS. FIRST APPEARS ON DAY 15 OF PREGNANCY IN ENDOMETRIUM OF COWS, REACHES A MAXIMUM ON DAY 18 AND REMAINS HIGH THROUGH DAY 26. [INDUCTION] BY TYPE I INTERFERONS. [SIMILARITY] CONTAINS 2 UBIQUITIN-LIKE DOMAINS.

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Abstract

L'invention concerne des méthodes de détermination de l'état de gestation d'un animal, suspecté d'être gravide, à l'aide d'essais combinés comprenant l'analyse de la progestérone et de la protéine à réaction croisée avec l'ubiquitine (UCRP), qui est également connue en tant que produit génétique stimulé par des interférons (ISG). La présente invention concerne des méthodes de diagnostic de la gestation à des stades très précoces. L'avantage de ce diagnostic précoce est que par identification des animaux qui ne sont pas gravides très peu de temps après l'accouplement, cela permet le réaccouplement rapide, réduisant ainsi au minimum la durée pendant laquelle l'animal est non gravide. L'invention concerne également des méthodes de prise de décision d'accouplement d'un animal. Lesdites méthodes permettent à un éleveur de prendre des décisions d'accouplement en fonction des niveaux d'UCRP et de progestérone détectés dans un échantillon biologique prélevé chez un animal suspecté d'être à des stades très précoces de gestation.
PCT/US2003/040192 2002-12-19 2003-12-17 Methode et moyen de detection precoce de gestation chez des animaux a l'aide d'essais combines WO2004059282A2 (fr)

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EP1546716A2 (fr) * 2002-05-02 2005-06-29 Aspenbio, Inc. Detection de la gravidite
CN1796998A (zh) * 2004-12-30 2006-07-05 万积成 用于奶牛早期妊娠诊断的试纸及其检测方法
US7713687B2 (en) 2000-11-29 2010-05-11 Xy, Inc. System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations
US7723116B2 (en) 2003-05-15 2010-05-25 Xy, Inc. Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US7758811B2 (en) 2003-03-28 2010-07-20 Inguran, Llc System for analyzing particles using multiple flow cytometry units
US7772005B1 (en) 1998-07-30 2010-08-10 Xy, Llc Method of establishing an equine artificial insemination sample
US7820425B2 (en) 1999-11-24 2010-10-26 Xy, Llc Method of cryopreserving selected sperm cells
US7833147B2 (en) 2004-07-22 2010-11-16 Inguran, LLC. Process for enriching a population of sperm cells
US7838210B2 (en) 2004-03-29 2010-11-23 Inguran, LLC. Sperm suspensions for sorting into X or Y chromosome-bearing enriched populations
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US8137967B2 (en) 2000-11-29 2012-03-20 Xy, Llc In-vitro fertilization systems with spermatozoa separated into X-chromosome and Y-chromosome bearing populations
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CN103163291A (zh) * 2013-03-09 2013-06-19 黑龙江八一农垦大学 一种试剂盒及操作方法
US9145590B2 (en) 2000-05-09 2015-09-29 Xy, Llc Methods and apparatus for high purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa
US9365822B2 (en) 1997-12-31 2016-06-14 Xy, Llc System and method for sorting cells
EP3220146A1 (fr) * 2016-03-18 2017-09-20 Stichting Wageningen Research Détection de durée correcte d'insémination par reconnaissance de motif de marqueur de grossesse multiple
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