IMMUNOLOGICAL APPROACH TO THE CHARACTERIZATION OF THE OUTER ACROSOMAL MEMBRANE OF BOAR SPERMATOZOA
IMMUNOLOGICAL APPROACH TO THE CHARACTERIZATION OF THE OUTER ACROSOMAL MEMBRANE OF BOAR SPERMATOZOA
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DOI：
10.1002/mrd.1120110205
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
SCHILL, WB
中科院分区：
文献类型：
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作者：
HINRICHSEN, AC;TOPFERPETERSEN, E;SCHILL, WB
用纯化的猪精子顶体外膜（OAM）制备抗血清，用猪肝和血清戊二醛交联免疫吸附剂吸附。抗血清的IgG级分通过（NH 4）2SO 4沉淀，然后通过离子交换色谱法纯化。间接免疫荧光显示明亮的荧光染色的顶体帽的猪精子和公牛和山羊精子的顶体在较小程度上孵育后，与抗OAM-IgG。免疫电镜进一步证实了抗体对OAM的特异性。将抗OAM-IgG与分离的OAM预孵育，完全消除了其反应性。当通过ELISA [酶联免疫吸附试验]进行检测时，抗OAM-IgG与猪、牛、山羊和人精子反应;与其他供试种属的精子相比，其与猪精子的结合活性显著更高。为了鉴定这种抗血清所识别的OAM抗原，分离的猪OAM用~ 3 H或~（125）I标记，并用温和的去污剂处理溶解。用抗OAM-IgG和含蛋白A的S.金黄色葡萄球菌和分离的抗原在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上进行分析。抗OAM-IgG可识别1种~ 3 H标记的高分子糖蛋白（270 kdalton）和4种~（125）I标记的低分子量多肽。
Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH4)2SO4 precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA [enzyme-linked immunosorbent assay], anti-OAM-IgG reacted with boar, bull, goat and human spermatozoa; its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens rcognized by this antiserum, the isolated boar OAM was labeled either with 3H or with 125I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the isolated antigens were analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Apparently, anti-OAM-IgG recognized 1 high molecular 3H-labeled glycoprotein (270 kdalton) and 4 125I-labeled polypeptides of lower MW of the boar OAM.