HORMONE DETECTION METHODS

Abstract: A method of predicting ovulation and a test kit is described which allows one to accurately predict the time of ovulation in an animal in advance thus permitting the highest rate of pregnancy to be achieved and at the same time minimising embryonic death. Also described is a test kit for detecting and quantifying a given reproductive hormone and a method of optimising fertility in animals.

Description: Hormone Detection Methods The present invention relates to fertility problems in animal particularly in horses, cows, sheep and pigs, and provides methods which animal fertility can be maximised. The fertility rate in thoroughbred horses is approximately 60%. Th poor level of fertility is caused by a number of factors includi the mating of mares too early in the breeding season when hormo levels may not be Optimal; limiting the use of the stallion so th the mare is not covered at the time of ovulation; hormone imbalanc in a mare as a result of a strenuous racing career and the mating stallions and mares which are not selected for fertility but rath for their racing performance. To obtain high conception rates, low reproductive wastage and hi foaling rates in the mare, it is necessary to cover the mare with highly fertile fresh stallion 12-24 hours prior to ovulation. Thi timing is critical to allow sperm to set up adequate reservoirs i the reproductive tract of the mare, and for final maturation o capacitation of sperm to take place in the uterus/oviducts befor ovulation. Although stallion spermatozoa can survive for 3-5 days i the reproductive tract of the mare, they have highest fertilizin capacity within 24 hours of covering. In contrast, the fertile lif span of the ovulated ovum is estimated to be of short duration ie 8-12 hours, and hence, the critical necessity of cover the mare a the optimum time before ovulation. However, because the duration of oestrus is variable, but generall of 4-7 days duration in the mare, and ovulation generally occur 24-48 prior to the end of oestrus, it is difficult to predic accurately during oestrus when the mare will ovulate, and hence whe to cover her. Following the regression of the corpus luteum an resulting decline in progesterone in the non-pregnant mare, th follicle continues to grow and oestradiol levels increase. The mar comes in season and the follicle increases in size by 2-3 mm per d and oestradiol levels rise concommitantly with the increased size the follicle. The follicle reaches a plateau in size and oestradi levels begin to decline 1-2 days before ovulation. As the follicl grows it probably produces increasing levels of a peptide hormo called inhibin; this hormone suppresses FSH in blood, there allowing only the single, but sometimes two, ovulatory follicles mature. Thus inhibin plays a key role in suppressing FSH duri oestrus to maintain the ovulation rate of mares at 1 in the majori of cases. As the ovulatory follicle develops during early oestru the increasing oestradiol is responsible for dilation of the cervi decreased uterine tone and exhibition of more intense signs oestrus prior to ovulation. In a recent study by Woods et a l (Ref. 4) it has been shown th the optimum time for insemination of mares is about one day prior t ovulation. The study showed that if insemination took place one d before ovulation 89% of inseminated mares became pregnant and those pregnancies 14% later exhibited embryonic death. Wh insemination took place on the day of ovulation there was a 5 pregnancy rate with a 34% embryonic death rate. Insemination the d after ovulation resulted in a 6% pregnancy rate and a 33% embryon death rate. Even the roost comprehensive rectal examination of mares which currently undertaken in which the ovaries are monitored with ultrasonic scanner, via the rectum, every three hours will only sh ovulation when it occurs. The above described study shows th insemination on that day will only result in a 52% pregnancy ra with a subsequent 34% embryonic death rate. It is known that oestradiol, or a major metabolite oestrone S0 increase daily during oestrus, plateau and decline before ovulatio Thus, this pattern of oestradiol could be used to time or predi ovulation accurately. However, this observation was of no ma practical benefit to the stud manager because of the lack of reliable, fast (results the same day) assay for oestradiol. It is thus an object of the present invention to be able accurately predict the time of ovulation in advance allowing t highest rate of pregnancy to be achieved and at the same ti minimising embryonic death. A further object is to provide a fas reliable assay to determine ovulation, particularly one whi accurately measures small amounts of reproductive hormones. A number of different techniques are currently used to optimi pregnancy. The mares are checked by swabbing for uterine a cervical infections and are then treated with antibiotics if they a found to be infected. The ovary can be manually manipulated by t veterinary surgeon to detect a large follicle thus determining th the mare is approaching ovulation. Ultrasonic scanning techniqu are used to detect pregnancy early on. Empirical treatments are al used in which combinations of hormones are administered to mar which gives wide variations in results and usually no pregnancy achieved. Finally, a combination of progesterone therapy and lighting regime are used in order to fool the mare into thinking th it is spring. The mares are subjected to eight weeks of increas exposure to electric light, the nutritional level is elevated a their heating is increased. This causes a small flow of hormones the mare and Follicle Stimulating Hormone (FSH) and Lutinizi Hormone (LH) to be produced by the anterior pituitary gland. Th 500mg of progesterone is given by injection. The progesterone sto the activity of the anterior pituitary gland and when t progesterone level falls off again a larger amount of FSH and LH a believed to be released. The mare then progresses into an estr cycle and comes in heat. This latter technique has a reasonab success rate compared with no intervention at all and 50% of mar subjected to this treatment may respond. The particular level of any hormone in the blood stream at any giv time taken together with the levels of any of the other hormon involved in pregnancy will cause a specific physiological effect o the reproductive process. It is therefore important to be able t access the level of a hormone in order to optimise pregnancy. If given hormone is not increasing in concentration in the bloodstrea as quickly as it should, it would be possible to immediatel administer an appropriate amount of the hormone to optimise th estrus cycle. In a normally cycling mare three to seven Graafian follicles wil develop in the ovary, under the influence of small levels of FSH an LH, to a size of greater than 18mm. This can be determined b ultra-sonic scanning. From day seven to day ten of the cycl selection of one or possibly two of these follicles takes place s that the follicle matures and increases in size. The trigger fo maturation of the follicle appears to be a surge in the concentratio of FSH in the bloodstream which takes place at about day seven of th cycle. It is believed that among the follicles developing at any on time, one follicle has a better capillary network which allows for a increased exposure to FSH in that particular follicle. As thi faster developing follicle grows it switches off the other follicle causing them to degenerate, probably by the presence of a loca hormone. Thus an appropriate level of FSH must be maintained betwee day seven and day ten in order to ensure release of an ovum. Oestradiol is a hormone which peaks 48 hours before ovulation, so a ability to determine oestradiol levels would allow for mating to b timed to the point of peak fertility. High levels of the hormon pro1actin in both stallions and mares leads to a high level o infertility, which can be treated with bromocryptine. Thus b accurately determining prolactin levels fertility can be enhanced Leutinizing hormone (LH) levels rise at approximately day fourteen o the cycle causing the Graafian follicle to rupture and leading t ovulation. Determination of LH levels permits mating to take plac at the time when conception is most likely to occur. It is thus a further object of the invention to provide a method t determine the actual level of any of the reproductive hormones in a animal thus allowing any imbalance in hormone levels to be correcte and also allowing mating to take place at a time which is most likel to lead to pregnancy. It has surprisingly been found that human antibodies directed agains the above described reproductive hormones are also capable o reacting with animal hormones, particularly equine hormones. For example, FSH occurs in mammals as a two sub-unit glycoprotein the two sub-units being non-covalently bound and referred to as th alpha and beta sub-units. Biological activity resides in the bet sub-unit. Although similar to an extent, FSH from different specie varies in its molecular size, amino-acid sequences and conformationa folding (references 1, 2 and 3). Comparing FSH from horses with that from humans shows: 1) the alpha sub-unit to be 10 amino acids shorter in equine FSH, 2) the alpha sub-unit to differ at 26 amino acid position between equine and human FSH and 3) the beta sub-unit contains 6 amino acid differences almos equally spaced along its 118 residues. The equine molecule is therefore different from the human molecule. It is particularly surprising to find cross-reactivity between th two types of FSH using a monoclonal antibody since monoclona antibodies are generally regarded to be specific and normall monoclonal antibodies are designed to avoid cross-reaction o decreased specificity. For a monoclonal antibody to be specific fo FSH it must be an antibody raised against the beta sub-unit or i must recognise part of the beta sub-unit together with part of th alpha sub-unit, since identical alpha sub-units are found in FSH Thyroid Stimulating Hormone (TSH), LH, and Chorionic Gonadotrophi (CG) within a single species. Anti-human FSH monoclonal antibodies are available from a number o commercial sources including Amersham International, Pic, England York Biologicals International, New York, U.S.A., and Immuno Search New Jersey, U.S.A. Anti-human prolactin monoclonal antibodies anti-human oestradiol polyclonal antibodies and anti-huma progesterone polyclonal antibodies are also commerically available from a number of sources including Amersham International Pic. According to the present invention there is provided a method of predicting ovulation in an animal comprising determining the level of a reproductive hormone in the bloodstream using an anti-reproductive hormone antibody. Ovulation may be predicted from a peak in oestradiol levels or from the lowest level of FSH. Preferably the antibody is an anti-human antibody. According to a further aspect of the present invention there is provided a method of detecting and quantifying a given reproductive hormone in animals in which an anti-human antibody directed against the given hormone is utilized in an immunometric assay. In a further aspect the invention relates to an anti-huma anti-reproductive-hormone antibody for use in the determination o reproductive hormone levels in animals. In a further aspect the invention provides a test kit for th detection of reproductive hormones in animals by immunometric assa utilizing an antibody as described above. In a particular embodimen the test kit comprising anti-oestradiol or anti-FSH antibodies may b used to predict ovulation. Many such assays are available such a chemiluminescence assays, enhanced luminescence assays radioimmunoassays and enzyme linked assays, all of which fall withi the scope of this invention. The anti-human antibody may be a monoclonal antibody. Th reproductive hormones detectable by this system include FSH progesterone, prolactin, and oestradiol. The animals may be horses sheep, cattle or pigs. The Amerlite (TM) system marketed by Amersham International Pic England is a non-radioactive immunoassay system for the detection o hormone levels in humans. The present invention also relates to non-radioactive immunoassay system of the same type as the Amerlit system for use in the detection of reproductive hormone levels i animals, particularly equines. In a particular embodiment the invention relates to a test kit fo the detection of FSH in equines by immunometric assay comprising a anti-human anti-FSH monoclonal antibody. Preferaby the monoclona antibody is a mouse anti-human antibody. The invention further relates to a method of optimising fertility i animals comprising:- (i) determining the level of at least one reproductive hormone i the animal by means of an immunometric assay which utilizes a anti-human, anti-reproductive hormone antibody, and (ii) adjusting the level of the hormone to a no al level either b administering hormone to the animal or by administering a drug t lower the hormone level. The invention also provides a method of maximising pregnancy i animals comprising:- (i) determining the level of a reproductive hormone, selected fro FSH and oestradiol, in the bloodstream of the animal by means of a immunometric assay which utilizes an anti-human, anti-reproductiv hormone antibody, and (ϋ) inseminating the animal within 48 hours of the occurance o either the peak level of oestradiol,or the lowest level of FSH, o both. Using the techniques of the present invention fertility and pregnanc can be maximised in a number of ways:- (1) Determine if Mare is Anoestrus: the anovulatory anoestuous mare can be accurately diagnosed by measurement of progesterone in the blood of mares taken at 4-7 day intervals. Low progesterone in a sequence of 4 samples taken at 4-7 day intervals is conclusive evidence that the mare is anoestrus. Presence of elevated progestrerone (greater than lng/ml) in one or more samples is evidence of the presence of a corpus luteu and hence that the mare has ovulated. This information is particularly valuable during the transition from the non-breeding to the breeding season. (2) Confirmation of Ovulation: following covering in oestrus, one or more blood samples can be taken, progesterone levels determined and a value of greater than Ing/ml is confirmation that the mare have ovulated. (3) Prolonged Dioestrus: a non-pregnant mare that fails to show oestrus 21-24 days after a previous oestrus (ie. foal heat) can be suspected to have prolonged maintenance of the life span of the corpus luteum (in non-pregnant mares it should repress after 14-16 days due to endogenous release of prostaglandin F2 alpha in the absence of the embryo). This can be confirmed by taking a blood sample after the failure to come in season 21-24 days after the previous oestrus and measuring progesterone in it. High levels of progesterone (greater than lng/ml) confirm that the mare is still in oestrus and appropriate veterinary treatment is required. (4) Primary Luteal Insufficiency: following covering, ovulation and conception in the mare, a small percentage of mares may secrete inadequate levels of progesterone to maintain viability of the embryo (require levels greater than 2ng/ml). Such mares could be identified by taking and measuring progesterone concentrations in 2-3 daily blood samples taken 6 or 7 days after ovulation. (5) Check function of Accessory Corpora: in the pregnant mare the endometrial cups are formed at 38-40 days of pregnancy and the produce equine chorionic gonadotrophin (eCG or formerly called PMSG) which is responsible for the formation of accessary corpora lutea. These then produce large quantities of progesterone essential for continued maintenance of pregnancy and viability of the embryo. I either insufficient eCG is produced or the accessory corpora lute are inadequate, the continuance of pregnancy might be jeopordized. Measurement of progesterone in 2-3 samples taken after day 60 coul help to confirm or not, that progesterone levels were adequate. Thi use, although not of general overall significance, could be importan in those mares prone to lose a pregnancy between days 45 and 150. The invention will now be described in greater detail in th following Examples. The FSH assay was carried out with the Amerlit System which is a complete immunoassay system comprising:- i) An analyser, which is an integrated luminescence reader with data reduction system, and microprocessor, (ii) A microtitre plate washer, (iii) An incubator/shaker, (iv) A pipetting station - either manual or robotic, (v) An interface to an IBM-compatible personal computer - with "RIA calc" data reduction facilities, (vi) An appropriate immunometric assay kit. In each case hormone levels in serum samples from six horses wer assayed. EXAMPLE 1 - FSH assay A commercially available kit for the detection of human FSH wa utilised in this example (available from Amersham Internationa Pic). The wells of the microtitre tray in the kit are coated wit sheep anti-FSH monoclonal antibody. The standard/control/sample wa added to the wells. A mouse monoclonal anti-human anti-FSH antibod which is linked to horseradish peroxidase was then added and allowe to equilibrate for one hour at 37°C. The unbound conjugate o monoclonal antibody and peroxidase was removed by aspiration an washing. A ' "signal" reagent (containing luminogenic substrates and peracid salt) with enhancer was then added into the wells. A comple reaction between peroxide, luminol and peroxides takes place in whic the peroxidase oxidises the luminogenic substrate and this oxidatio reaction causes the emission of light. The enhancer is a substituted phenol which increases the level o light produced and prolongs its emission. Because the light level from enhanced luminescence are of high intensity, the reaction can b optimized to give a continuous output of light rather than a flash. The light signal can be measured without critical timing and th signal can be re-measured if desired. The evolved light is measured at 2 to 20 minutes post-addition of signal and enhancer. The amount of conjugate bound to the wells is directly proportional to the concentration of FSH present in the standard/control/sample. The results of this test are shown in Table 1. It is also possible to determine FSH levels in a sample using a non-competitive "sandwich-type" immunometric assay system. EXAMPLE 2 - Oestradiol assay Oestradiol levels were determined by a competitive radioimmunoassay which uses donkey anti-rabbit antibody and rabbit anti-human anti-oestradiol polyclonal antibody. Radioactively labelled oestradiol and cold standard/controls/sample are then added into the assay system where they compete for binding sites on the bound anti-oestradiol antibody. The amount of radioactive label bound is inversely proportional to the amount of oestradiol in the sample. The oestradiol assay utilises a rabbit anti-oestradiol polyclonal antiserum which allows for some cross-reactivity with oestradiol-3-sulphate, oestrone and oestriol. The results are shown in Table 1. EXAMPLE 3 Progesterone and prolactin levels were also determined using polyclonal and monoclonal anti-human antibodies respectively. The results are shown in Table 1. TABLE 1 FSH mlU/ml (MONOCLONAL) Amerlite controls:- "Tru-value" controls Low = 10.3 Low = 5.7 Medium = 25.0 Medium = 28.3 High = 50.2 High = 70.2 Patient I = 1.6 Pregnant Mare Patient II = 3.8 II Patient III = 14.5 Cycling Mare Patient IV = 14.0 II Patient V = 13.5 II Patient VI = 36.9 Gelding PROLACTIN mlU/L (MONOCLONAL) Amerlite controls:- "Tru-value" controls Low = 12.2 Medium = 1817 High = 5080 Pregnant Mare (milk "comming-in") Cycling Mare Gelding * Results of serum samples differed from mean results of plasma sampl OESTRADIOL omol/L (POLYCLONAL) RSL Controls Wein Controls Low = 286 Low = 293 Medium = 619 Medium = 2621 High = 1841 High = 5182 Patient I = 10938 * Pregnant Mare Patient II = 11889 * II Patient III = 240** Cycling Mare Patient IV = 520** II Patient V = 240** Cycling Mare Patient VI = 325 Gelding * Cross-reaction with oestriol? ** Levels compatible with mares in oestrous phase of cycle correlates with FSH results. PROGESTERONE nmol/L (POLYCLONAL) Amerlite Controls RSL Controls Wein Controls Low = 0.92 Low = 2.4 Medium = 27.0 Medium = 21.4 High = 72.8 High = 51.3 Pregnant Mare II Cycling Mare II II Gelding N.D. = Not Detected. Example 4 A trial was conducted on a total of 112 mares in which oestradiol a FSH levels in peripheral blood were determined daily over the period which the mare was in heat, by the techniques discussed in Examples and 2. The mares were also examined by a clinician using convention techniques for determining ovulation e.g. rectal examination f development of the size softness and contour of the follicles, a relaxation of the cervix. Ultrasonic scanning was also used to measu follicle growth and to determine when ovulation had occured based the disappearance of the pre-ovulatory follicle and the presence of corpus haemoragium in its place the next day. A comparison of peak hormone levels with time of ovulation showed th 84.6% of mares ovulated within 48 hours of the oestradiol peak. In 6 of mares the FSH level fell to its lowest point at the same time as t oestradiol level peaked. The assay proved to be rapid and accurate and has the advantage that does not involve the use of radioisotopes or other hazardous chemicals REFERENCES Fujiki Y., Rathnam P. and Saxena B.B., Amino acid sequence of the subunit of the Follicle Stimulating Hormone from equine pituita glands, J.Biol Chem. 253.5363-5368, 1978. Hojo H. and Ryan R.J., Monoclonal antibodies against human Follic Stimulating Hormone, Endochrinology, 2428-2434, 1985. Rathnam P., Fuijiki Y., Landefeld T.D. and Saxena B.B., Isolation a amino acid sequence of the alpha subunit of Follicle Stimulati Hormone from equine pituitary glands, J. Biol. Chem. 253.5355-536 1978. Woods J., Bergfelt D.R. and Ginter O.J., Effects of time insemination relative to ovulation on pregnancy rate and embryonic lo rate in mares. Equine Veterinary Journal. 22 (6) 410-415, 1990.

Claims: CLAIMS 1. A method of predicting ovulation in an animal comprisin determining the level of a reproductive hormone in the bloodstream b an immunometric assay using an anti-reproductive hormone antibod wherein the anti-reproductive antibody is an anti-human antibody. 2. A method as claimed in claim 1 wherein the peak oestradio level is determined. 3. A method as claimed in claim 1 wherein the lowest level of FS is determined. 4. A method of detecting and quantifying a given reproductiv hormone in an animal in which an anti-human antibody directed agains the given hormone is utilized in an immunometric assay. 5. A method as claimed in any previous claim wherein the antibo is a monoclonal antibody. 6. A method as claimed in any preceding claim wherein the antibo is a mouse antibody. 7. A method as claimed in claim 4 wherein the reproductive hormo is selected from oestradiol, follicle stimulating hormone, progestero or prolactin. 8. A method as claimed in any preceding claim wherein the anim is an equine. 9. A test kit for the detection of reproductive hormones in animal by immunometric assay comprising an anti-human, ant reproductive hormone antibody. 10. A test kit as claimed in claim 9 wherein the reproducti hormone is selected from oestradiol, follicle stimulating hormon progesterone or prolactin. 11. A test kit for the prediction of ovulation by immunometr assay comprising an anti-human, anti-oestradiol or anti-FSH antibody. 12. A test kit as claimed in any of claims 9 to 11 wherein t antibody is a monoclonal antibody. 13. A test kit as claimed in claim 11 or 12 wherein the antibody a mouse antibody. 14. A method of optimising fertility in animals comprising:- (i) determining the level of at least one reproductive hormone the animal by means of an immunometric assay which utilizes anti-human, anti-reproductive hormone antibody, and (ii) adjusting the level of the hormone to a normal level either administering hormone to the animal or by administering a drug to low the hormone level. 15. A method of maximising pregnancy in animals comprising:- (i) determining the level of a reproductive hormone, selected fr FSH and oestradiol, in the bloodstream of the animal by means of immunometric assay which utilizes an anti-human, anti-reproducti hormone antibody, and (ii) inseminating the animal within 48 hours of the occurance either the peak level of oestradiol or the lowest level of FSH, or bot 16. An anti-human, anti reproductive-hormone antibody for use i the determination of reproductive hormone levels in animals. 17. An antibody as claimed in claim 16 wherein the reproducti hormone is selected from oestradiol, follicle stimulating hormon progesterone or prolactin.

Inventors: O'CONNOR MICHAEL, O'CONNOR, MICHAEL
Applicants: ENFER TECH LTD, NEWBY MARTIN JOHN, ENFER TECHNOLOGY LIMITED, NEWBY, MARTIN, JOHN
Application year: 1991
Main classification: G01N33
IPC codes: A61B10, A61D17, C07K16, G01N33