Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-17
2004-09-07
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007910, C435S007920, C435S007930, C435S007940, C435S007950, C436S510000, C436S514000, C436S518000, C436S525000, C436S526000, C436S065000, C436S814000, C436S815000, C436S817000
Reexamination Certificate
active
06787324
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of detecting conception and/or implantation in animals, including humans, and apparati therefor.
BACKGROUND OF THE INVENTION
It has been a long-sought goal of physicians and veterinarians to have reliable diagnostic markers for conception, implantation and viable pregnancies, to help manage treatment of infertility and early pregnancy treatment. In humans, early pregnancy diagnosis based on placental protein markers is only routinely relied upon at 4 weeks after conception, and ultrasonic analysis is only reliably positive at 7-8 weeks gestation.
In the livestock industry, it is important to be able to identify animals that have not successfully conceived following breeding. For example, in the cattle industry at the present time, there is no way to identify such animals before 35-40 days after breeding, and the identification must be made by a veterinarian using palpation. Alternatively, ultrasound analysis can detect a developing embryo at 21 days. Veterinarian palpation is by far the most commonly used method, costing approximately $4 to $10 per test. The cost for an ultrasound analysis is prohibitive for routine farm management. In addition to the costs of these tests, the farmer suffers an additional and significant financial loss in having cows that have been bred but have not conceived, also referred to as “open” cows. The “open” cow costs the farmer an additional $4 to $10 dollars per day. The better milk producers are the hardest cows to breed, so while they are “open” the loss is even greater. Less than 50% of cows conceive on the first breeding. Due to this fact, the usual breeding program allows for 2½ semen straws per cow. If the time interval during which a cow is “open” can be shortened to days instead of months, this would substantially increase the overall calving rates.
A factor, named early pregnancy factor (EPF) or more recently immunosuppressive early pregnancy factor (ISEPF), has been detected in animals using a bioassay, and it is thought to be responsible for suppressing the maternal immune response against the embryo/fetus. Despite the demonstration of the activity through a bioassay, the literature presents several different MW forms for ISEPF. In mice, Clarke et al. (Clin Exp. Immunol. 32:318, 1978) reported an EPF of approximately 180,000 kD. In sheep, Clarke et al (J. Reprod. Immunol. 1980 Vol. 2:151) described the existence of multiple forms of EPF, including 20 kD, 50 kD, and 250-350 kD forms. In a 1987 paper from the same laboratory, Clarke et al describe the purification of a 12 kD EPF from the placenta of 12 weeks pregnant sheep (J. Reprod. Immunol. 1987 10:133-156). Cavanaugh described the purification of a 21 kD EPF from cultured ovaries and oviducts of mice, which is composed of three subunits, 10.5, 7.2 and 3.4 kD in size (J. Reprod. Fertil. 71:581, 1984). The factor has most recently been described as a glycoprotein of high molecular weight (Threlfall, 1993), but prior to this invention, a functionally pure preparation was not known.
An indirect bioassay for the ISEPF utilizes an in vitro, rosette inhibition assay described by Bach and Antoine (Nature 217:658 1968), which measures the ability of ISEPF to enhance the inhibition of rosette formation between T cells and heterologous erythrocytes induced by anti-lymphocyte serum (ALS). Both molecular weight components must be present to detect ISEPF in the rosette assay. It has been postulated that the ALS sterically hinders the binding of the erythrocytes in the assay; ISEPF enhances the inhibition by saturating some binding sites on the lymphocytes (Smart, Y C et al., Fertil. & Steril. 35:397, 1981). ISEPF has been found in the mouse (Morton et al., Nature 249:459 1974), rat (Heywood, L H et al. Australian Soc. for Reprod. Biol. 1979), human Morton, H. et al., Lancet 394 1977), sheep (Morton, H. et al. Res. in Vet. Sci. 26:261 1979), pig (Grewal, A S et al. Australian Soc. for Reprod. Biol. 1981), and cattle (Nancarrow et al. J. Reprod. & Fert. 57:385 1979). Noonan et al (Nature 278:629 and 649 1979) have described ISEPF as species non-specific.
Given the appearance of ISEPF very soon after mating, it is possible that ISEPF could be an excellent early marker for conception in animals. However, the rosette inhibition assay is technically difficult to perform, time-consuming, cumbersome and subject to numerous false-positive readings (Sinosich et al., 1985). To develop an ISEPF assay that is reproducible and not subject to a large number of false-positive signals, a substantially pure preparation of ISEPF is required. Prior to this invention, no protocols for the complete purification of a high molecular weight ISEPF have been reported.
There remains a need for a reliable assay to detect pregnancy as early as possible after conception and further to detect spontaneous abortion.
There is a need to be able to breed animals and determine, within 12-48 hours, whether the breeding has resulted in conception. In cattle, as an example, such non-conceiving cows could be recycled with injections of prostaglandin and inseminated again without the loss of thirty days. There is further a need to be able to enhance the ability of elite cows to implant at a higher rate.
SUMMARY OF THE INVENTION
The present invention provides a purified factor, herein referred to as the “early conception factor” or ECF, antibodies specific for ECF, and kits and apparatuses for detecting the presence or absence of ECF in fluid or tissue samples taken from animals. Methods for detecting conception within 12-48 hours of breeding/mating are described. Methods for detecting fetal death following conception and implantation are also provided. Means for enhancing embryonic implantation utilizing the ISEPF and the anti-ISEPF antibodies of this invention are also provided.
REFERENCES:
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Morton et al., “Ovum Factor and Early Pregnancy Factor.”Curr. Topics Dev. Biol. 23:73-92 (1987).
Trapani et al., “A re-examination of the association of ‘early pregnancy factor’ activity with fractions of heterogeneous molecular weight distribution in pregnancy sera.”Early Pregnancy: Biology and Medicine3(4):312-322 (Dec. 1997).
Zuo et al., “Isolation and Characterization of Early Pregnancy Factor.”Chin. Med. Sci. J. 9(1):34-37 (1994).
Quinn et al., “Monoclonal antibodies to early pregnancy factor perturb tumour cell growth,”Clin. Exp. Immunol. 80(1):100-108 (Apr. 1, 1990).
W.J.Sinosich, et al. “Placental Proteins in the Diagnosis and Evaluation of the Elusive Early Pregnancy”Obstetrical and Gynecological Survey, vol. 40. No. 5, 1985, pp. 273-282.
B.E. Rolfe, et al. “Early Pregnancy factor in an immunosuppressive contaminant of commercial preparations of human chorionic gonadotropgin”Clinical Exp. Immunology, 1983, vol. 51: pp. 45-52.
F.M. Clarke, et al. “Partial Characterisation of Early Pregnancy Factor in the Sheep”,Journal of Reproductive Immunology2 (1980) pp. 151-162.
F.M. Clarke, et al. “Detection and separation of two serum factors responsible for depression of lymphocyte activity in pregnancy”Clinical Exp. Immunology1978, vol. 32:pp. 318-323.
F.P. Noonan, et al. “Early Pregnancy Factor is Immunosuppressive”Naturevol. 278, 1979, pp. 649-651.
Y. Cheng Smart, et al.“Early Pregnancy Factor: Its Role in Mammalian Reproduction-Research Review”Pregnancy and Sterility, vol. 35. No 4. Apr. 1981, pp. 397-402.
A.C. Cavanagh,“Production in vitro of mouse early pregnancy factor and purification to homogeneity”J. Reprod. Fert. (1984) 71, pp. 581-592.
F.M. Clarke, et al.“Early Pregnancy factor: Large scale isolation of rosette inhibition test-active polypeptides from ovine placental extracts”Journal of Reproductive Immunology, 10 (1987) 133-156.
W.R. Threlfall,(A) “Early Conception Assay Application To Diary Cow Pregnancy Determination”Soc. Theriogenol. Ann Conf., Aug. 12-14, 1993.
W.R. Threlfall,(B)“Immu
Jordan John Douglas
Jordan Nancy Tommye
Chin Christopher L.
Concepto Diagnostics, Inc.
Needle & Rosenberg P.C.
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