Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development
Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development
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DOI：
10.1093/humrep/den088
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发表时间：
2008-06-01
影响因子：
6.1
通讯作者：
Naz, R. K.
中科院分区：
文献类型：
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作者：
Samuel, A. S.;Naz, R. K.
背景：避孕疫苗可以为当前避孕方法提供有价值的替代方案。我们在这里描述了用于免疫避孕的精子反应性人单链可变片段（scFv）抗体的开发，该抗体具有明确的精子特异性。方法：用人精子抗原在体外激活抗精子抗体阳性的免疫不育和输精管切除男性的外周血白细胞（PBL），制备互补DNA并使用基于免疫球蛋白重链和轻链所有可变区的引物进行PCR扩增。将 scFv 库克隆到 pCANTAB5E 载体中以创建人 scFv 抗体库。结果：针对特定精子抗原对文库进行淘选，产生了几个克隆，并选择了四个最强的反应性进行进一步分析。这些克隆具有带有独特互补决定区的新颖序列。表达、纯化 ScFv 抗体并分析其对人类精子的反应性及其对人类精子功能的影响。 AFA-1 和 FAB-7 scFv 抗体均与受精抗原 1 抗原发生反应，但针对不同的表位。 YLP20 抗体与预期的 48 +/- 5 kDa 人类精子蛋白发生反应。第四种抗体 AS16 与 18 kDa 精子蛋白发生反应，似乎是引起精子凝集的小鼠单克隆重组抗精子抗体的人类同源物。所有这些抗体都会抑制人类精子的功能。结论：这是第一项报告使用噬菌体展示技术获得具有确定抗原特异性的抗精子 scFv 抗体的研究。这些抗体将在新型免疫避孕药的开发以及免疫不孕症的特异性诊断中找到临床应用。
BACKGROUND: Contraceptive vaccines can provide valuable alternatives to current methods of contraception. We describe here the development of sperm-reactive human single chain variable fragment (scFv) antibodies of defined sperm specificity for immunocontraception. METHODS: Peripheral blood leukocytes (PBL) from antisperm antibody-positive immunoinfertile and vasectomized men were activated with human sperm antigens in vitro, and the complementary DNA prepared and PCR-amplified using primers based on all the variable regions of heavy and light chains of immunoglobulins. The scFv repertoire was cloned into pCANTAB5E vector to create a human scFv antibody library. RESULTS: Panning of the library against specific sperm antigens yielded several clones, and the four strongest reactive were selected for further analysis. These clones had novel sequences with unique complementarity-determining regions. ScFv antibodies were expressed, purified and analyzed for human sperm reactivity and effect on human sperm function. AFA-1 and FAB-7 scFv antibodies both reacted with fertilization antigen-1 antigen, but against different epitopes. YLP20 antibody reacted with the expected human sperm protein of 48 +/- 5 kDa. The fourth antibody, AS16, reacted with an 18 kDa sperm protein and seems to be a human homologue of the mouse monoclonal recombinant antisperm antibody that causes sperm agglutination. All these antibodies inhibited human sperm function. CONCLUSIONS: This is the first study to report the use of phage display technology to obtain antisperm scFv antibodies of defined antigen specificity. These antibodies will find clinical applications in the development of novel immunocontraceptives, and specific diagnostics for immunoinfertility.