Production of human monoclona. antibody to spermatozoa and its application for purification of the corresponding sperm antigen.

将小鼠骨髓瘤细胞(NS-1)与具有精子固定(SI)活性的不孕症妇女外周血淋巴细胞融合，建立了具有较强的精子固定(SI)活性的人-鼠杂交瘤。用同一供体的淋巴细胞在不同的时间建立了H6-oC4、4A1、4A4三种节瘤细胞，经鉴定为同一克隆。它们产生相同的人IgM；lt；lambda&gt；具有很强的SI和精子凝集(SA)活性，并在人类射出的精子上表现出相同的免疫结合模式。用从H6-3C4杂交瘤中分离的免疫球蛋白基因(VH-3C4，V&lt；lambda&gt；-3C4)进行的Northern和Southern杂交分析也显示出类似的模式。单抗H6-3C4不仅能与人精子反应，还能与人精浆反应。提示相应的抗原为精子包被的热休克蛋白抗原。当热休克蛋白被分馏时，…用饱和硫酸铵沉淀后，再经Sephacry1 S-300凝胶过滤，相应的抗原在胞体部分(&gt；M.W.670KD)中得到洗脱，而空泡部分经十二烷基硫酸钠-PAGE和Western blotting后，在约20KD的宽带内检测到抗原性。抗原性对煮沸和酶处理有较强的抵抗力，但用高碘酸或三氟甲烷磺酸处理后，抗原性明显减弱。同样，神经氨酸酶被-lt；β&gt；-半乳糖苷酶和&lt；β&gt；-N-乙酰氨基葡糖苷酶污染后，抗原活性显著降低。这些结果表明，与Mad H6-3C4相应的抗原表位的形成可能与碳水化合物部分而不是多肽有更强的相关性。在用SI-ADS检测人精子与人精子的竞争结合抑制试验中，除供者外，仅有1例患者表现出明显抑制作用。提示H6-3C4相应的SI-Ab产生克隆在不孕症妇女中似乎很少见。较少

Human-mouse heterohybridomas producing human monoclonal antibodies(Mads) with a strong sperm immobilizing(SI) activity were established by fusing mouse myeloma cells(NS-1) with peripheral blood 1ymphocytes from a sterile women with SI antibody. Thbridomas(H6-oC4, 4A1, 4A4) Which were established in different occasions by using the same donor 1ymphocytes were proved to be the same clone. They produced the same human IgM<lambda> with strong SI and sperm agglutinating (SA) activities,and showed the same immunostaing patterns on ejaculated human spermatozoa. The competitive binding inhibition by these antibodies to spermatozoa, and Northern and Southern blot analyses using immunoglobulin genes (VH-3C4, V<lambda>-3C4) isolated from H6-3C4 hybridoma showed also the similar patterns. The Mab H6-3C4 reacted not only to ejaculated human spermatozoa but also human seminal plasma(HSP). It implied that the corresponding antigen was the sperm-coating HSP antigen. When HSP proreins were fractionated … More by ge1 filtration on Sephacry1 S-300 after precipitation with saturated ammonium sulfate, the corresponding antigen was eluted in the boid fraction(>M.W. 670KD),but when the void fraction was subjected to SDS-PAGE and followed by western blotting,the antigenecity was detected in a broad band arond 20KD. The antigenecity was relativery resistant to boiling and pronase treatments but markedly diminished by treating with periodic acid or trifluoromethane sulfonic acid. Similarly,the treatment with neuraminidase which was contaminated with <beta>-galactosidase and <beta>-N-acetylagucosaminidase markedly reduced the antigen activities. These results suggested that carbohydrate moieties rather than peptides might be more strongly related to comformation of the antigen epitope correesponding to the Mad H6-3C4. In the competitive binding inhibition assay of^<125>I-H6-3C4 Mab to human spermatozoa by a number of patient's sera with SI-Ads, only one patient excluding the donor of lymphocytes for cell fusion showed a significant inhibition. It suggested that the SI-Ab producing clone corresponding to H6-3C4 seems to be rare in infertile women. Less