DELINEATION OF EPITOPES ON PORCINE ZONA-PELLUCIDA RELEVANT FOR BINDING OF SPERM TO OOCYTE USING MONOCLONAL-ANTIBODIES

被引:25
作者
BAGAVANT, H
YUREWICZ, EC
SACCO, AG
TALWAR, GP
GUPTA, SK
机构
[1] NATL INST IMMUNOL,GAMETE ANTIGEN LAB,JNU COMPLEX,NEW DELHI 110067,INDIA
[2] WAYNE STATE UNIV,SCH MED,CS MOTT CTR HUMAN GROWTH & DEV,DEPT OBSTET & GYNAECOL,DETROIT,MI 48201
关键词
D O I
10.1016/0165-0378(93)90047-L
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Seven monoclonal antibodies (MAs) generated against porcine zona pellucida glycoprotein, ZP3 (comprising both ZP3alpha and ZP3beta) were characterized for their specificities to ZP3alpha (MA 7 and MA 28) or ZP3beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3alpha and MA 30 for ZP3beta. All the antibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.
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页码:265 / 279
页数:15
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