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

人单克隆抗体的生产。

• 批准号: 61570815
• 负责人: KOYAMA Koji
• 金额: $ 1.34万
• 依托单位: Hyogo College of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61570815/
• 关键词: Immunological sterility; Sperm immobilizing antibody; Cell fusion; Human-mouse heterohybridome; ヒト型精子不動化モノクローナル抗体; 精子不動化関連抗原; 糖鎖抗原; 避妊ワクチン; ヒト-マウスハイブリドーマ; ヒト単一クローン抗精子抗体; 不妊症; 免疫学的避妊ワクチン

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

通过将小鼠骨髓瘤细胞（NS-1）与具有SI抗体的不育妇女外周血淋巴细胞融合，建立了产生具有强精子固定化活性的人-小鼠异杂交瘤。用同一供体淋巴细胞在不同场合建立的胸腺瘤（H6-oC4, 4A1, 4A4）被证明是同一克隆。它们产生了相同的人IgM< λ >，具有较强的SI和精子凝集（SA）活性，并在射精的人精子上显示出相同的免疫染色模式。这些抗体对精子的竞争性结合抑制作用，以及对H6-3C4杂交瘤中分离的免疫球蛋白基因（VH-3C4， V<lambda>-3C4）的Northern和Southern blot分析也显示了类似的模式。该单抗H6-3C4不仅对人射精精子有反应，而且对人精浆（HSP）也有反应。提示对应抗原为精子包被HSP抗原。HSP原蛋白经饱和硫酸铵沉淀后，在Sephacry1 S-300上进行ge1过滤，在固体馏分（>M.W.）中洗脱670KD)，但当将空洞部分进行SDS-PAGE和随后进行western blotting时，抗原在20KD左右的宽波段检测到。抗原性对沸煮和酶处理具有相对的抗性，但经周期酸或三氟甲烷磺酸处理后，抗原性明显降低。同样，被< β >-半乳糖苷酶和< β >- n -乙酰氨基糖苷酶污染的神经氨酸酶处理显著降低了抗原活性。这些结果表明，与Mad H6-3C4对应的抗原表位构象密切相关的可能是碳水化合物部分而不是肽段。在许多SI-Ads患者血清中，^<125>I-H6-3C4单抗与人精子的竞争性结合抑制实验中，除了用于细胞融合的淋巴细胞供体外，只有一名患者表现出明显的抑制作用。这表明产生SI-Ab的H6-3C4克隆在不育女性中似乎很少见。少

项目成果

山崎則行他: 産婦進歩. 39. 567 (1987)

Noriyuki Yamazaki 等人：妇产科进展 39. 567 (1987)

Shinzo Isojima et al: Am.J.Reprod.Immunol.Microbiol.10. 90-92 (1986)

Shinzo Isojima 等人：Am.J.Reprod.Immunol.Microbiol.10。

繁田実 他: 日本免疫学会総会・学術集会記録. 16. 263- (1986)

Minoru Shigeta 等：日本免疫学会会员大会和学术会议记录 16. 263- (1986)。

S,Kyurkchiev et al: Immunology. 57. 489-492 (1986)

S，Kyurkchiev 等人：免疫学。

亀田絹他: 産婦進歩. 39. 563-566 (1987)

Kinu Kameda 等人：产科进展 39. 563-566 (1987)