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DEVELOPMENT OF MONOCLONAL AND POLYCLONAL ANTIBODIES AGAINST BOVINE PREGNANCY-ASSOCIATED GLYCOPROTEINS (PAG) FOR USE AS REAGENTS IN LOCALIZATION OF PAG EXPRESSION AND FOR PREGNANCY DETECTION.
Avalle, Mary1, Hook, Reuel2, McClain, April1, Green, Jonathan1, Mathialagan, Nagappan3, Egodage, Kamal 3, Roberts, Robert1,2, 1 2 3
ABSTRACT- Pregnancy-associated glycoproteins (PAGs) constitute a group of proteins within the aspartic proteinase superfamily that are secreted by the trophoblast of the ruminant placenta. Some are produced by the invasive binucleated cells, while others are expressed throughout the trophectoderm. PAGs also differ in their temporal expression patterns during pregnancy. The goal of this work has been to produce serological agents directed against both individual and groups of PAGs that would a) provide further information on PAG expression in placenta and b) allow development of a non-isotopic pregnancy test for cattle. Rabbit polyclonal antisera were raised against PAG preparations that had been subfractionated according to their relative affinities for an aspartic proteinase inhibitor, pepstatin A. Murine monoclonal antibodies were generated toward affinity-purified bovine PAGs, isolated from d24-34 trophoblast cultures and d80 cotyledonary extracts. The resulting monoclonal and polyclonal antibodies exhibited distinguishable patterns of staining of placental extracts on western blots, suggesting that they were directed toward different epitopes. Fifty anti-PAG hybridoma lines were propagated. Four monoclonal antibodies, recognizing early PAGs, were selected for ELISA assays. The most promising assay to date used a mixture of trapping monoclonal antibodies (A3, J2, L4) and a secondary anti-PAG polyclonal antibody. Antigen was detected in >77% of pregnant cows and heifers by day 26, and in 100% by day 30. PAG concentrations rose gradually, peaking just prior to parturition, and then declined rapidly, becoming undetectable by 5 weeks postpartum. This test, although not yet optimized, avoids the major problem in the existing assays for boPAG-1 (PSP-B) where cross-reacting antigen remains elevated into the postpartum re-breeding period. Supported by USDA grant 96-35203-3257 and funding from Monsanto Co.
KEY WORDS: pregnancy-associated glycoprotein, antibody, ELISA
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