D'Cruz O J, Haas G G, Lambert H
Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
J Immunol. 1993 Jul 15;151(2):1062-74.
Target Ag recognized by clinically important C-fixing anti-sperm antibodies (ASA) were identified by indirect immunoprecipitation after incubation of biotinylated, motile sperm with ASA-positive sera from autoimmune, isoimmune, and vasectomized patients. This method assessed native protein thus permitting analysis of conformation-dependent membrane Ag that would be missed by previously reported Ag identification techniques. Sera from 35 ASA-positive and 10 ASA-negative patients were exposed to capacitated, surface-biotinylated motile sperm. After detergent-extraction of the membrane fraction, the immune complexes were precipitated with protein G-agarose. The Ag were eluted under reducing conditions, electrophoresed on SDS-10% PAGE, electroblotted onto nitrocellulose membranes, and visualized by the biotin/avidin-peroxidase detection method. By this method, only high titered ASA-positive sera demonstrated variable reactivity restricted to a total of eight to nine protein bands. Individual sera revealed a differential reactivity to a constant set of protein bands (63/61, 58/56, 29, and 21/19 kDa) and a preferential reactivity to either 45-, 43-, or 41-kDa protein Ag that were patient specific. None of these bands were precipitable by ASA-negative sera. ASA directed against the 63/61-kDa and 58/56-kDa polypeptides were found in 85% of immunoprecipitable sera; the 29-kDa was detectable with 70% of sera. Immunoreactivity to the 45-kDa to 41-kDa bands was detectable with 30% of the immunoprecipitable sera. Although the pattern of Ag recognized by ASA from autoimmune, isoimmune, and vasectomized patients was similar, our results indicate that in addition to the recognition of common human sperm surface determinants, distinct sperm surface epitopes are also recognized as foreign by the patient's immune system. Sera that immunoprecipitated the predominant monomeric 29-kDa or the dimeric 58/56-kDa Ag when preincubated with capacitated sperm markedly inhibited sperm binding and penetration to the human zona when compared with ASA-negative serum controls. It is suggested that C-fixing ASA directed to these surface determinants may account for some of the clinical manifestations of ASA-mediated infertility.
通过将生物素化的活动精子与自身免疫性、同种免疫性和输精管切除患者的抗精子抗体（ASA）阳性血清孵育后，采用间接免疫沉淀法鉴定了临床重要的补体结合抗精子抗体所识别的靶抗原。该方法评估天然蛋白质，从而能够分析依赖构象的膜抗原，而这是先前报道的抗原鉴定技术所遗漏的。将35例ASA阳性患者和10例ASA阴性患者的血清与获能的、表面生物素化的活动精子接触。在对膜部分进行去污剂提取后，用蛋白G琼脂糖沉淀免疫复合物。在还原条件下洗脱抗原，在SDS - 10%聚丙烯酰胺凝胶上进行电泳，电转移到硝酸纤维素膜上，并用生物素/抗生物素蛋白 - 过氧化物酶检测方法进行可视化。通过这种方法，只有高滴度的ASA阳性血清表现出可变的反应性，局限于总共八到九条蛋白带。个体血清对一组恒定的蛋白带（63/61、58/56、29和21/19 kDa）表现出不同的反应性，并且对45 - 、43 - 或41 - kDa的蛋白抗原有优先反应性，这些是患者特异性的。这些带中没有一条能被ASA阴性血清沉淀。在85%的可免疫沉淀血清中发现了针对63/61 - kDa和58/56 - kDa多肽的ASA；70%的血清可检测到29 - kDa。30%的可免疫沉淀血清可检测到对45 - kDa至41 - kDa条带的免疫反应性。尽管自身免疫性、同种免疫性和输精管切除患者的ASA所识别抗原模式相似，但我们的结果表明，除了识别常见的人类精子表面决定簇外，患者的免疫系统也将独特的精子表面表位识别为外来物。与ASA阴性血清对照相比，在与获能精子预孵育时免疫沉淀主要单体29 - kDa或二聚体58/56 - kDa抗原的血清显著抑制精子与人透明带的结合和穿透。提示针对这些表面决定簇的补体结合ASA可能是ASA介导的不孕症某些临床表现的原因。
