1987 Fiscal Year Final Research Report Summary
Production of human monoclona. antibody to spermatozoa and its application for purification of the corresponding sperm antigen.
| Project/Area Number |
61570815
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Hyogo College of Medicine |
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Principal Investigator |
KOYAMA Koji Hyogo College of Medicine, 医学部, 助教授 (00068496)
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| Co-Investigator(Kenkyū-buntansha) |
SHIGETA Minoru Hyogo College of Medicine, 医学部, 助手 (80122315)
TSUJI Yoshiyuki Hyogo College of Medicine, 医学部, 助手 (60148658)
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| Project Period (FY) |
1986 – 1987
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| Keywords | 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 |
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| Research Abstract |
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
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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
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