人抑制素α亚基单克隆抗体的制备和鉴定

Preparation and identification of human inhibin α subunit monoclonal antibody

目的：建立分泌抗人抑制素α亚基３７ － ６５ 片段单克隆抗体（ 单抗） 细胞株，并对单抗进行免疫学鉴定。方法：以猪卵泡液粗提物为免疫原，免疫ＢＡＬＢ／ｃ 小鼠，取其脾细胞与ＮＳ１ 小鼠骨髓瘤细胞融合。杂交瘤细胞用人工合成的人抑制素α亚基３７ －６５ 片段包被，间接ＥＬＩＳＡ法筛选。结果：获得稳定分泌抗人抑制素α亚基３７ － ６５ 片段单抗细胞３ 株，ＥＬＩＳＡ 检测腹水效价为１×１０－ ５～１×１０ －３ ，与α亚基１ －３２ 及其他多肽激素无交叉反应，３ 株识别同一抗原位点。相对亲和力为ⅠＡ５ ＞ Ⅳ３Ｃ＞ Ⅴ５Ｇ。腹水中的抗体能明显地被人卵泡液和猪卵泡液和α（３７ － ６４）中和。结论：３ 株细胞有特异性、敏感性和实用性。

Aims:To establish anti human inhibin α subunit 37-65 fragment antibody cell line,and to perform immunological identification to the antibody. Methods: The extracted substance of porcine follicular fluid was used as immunogen. The spleen cells of BALB/c mice that had been immuned were fused with mouse myeloma (NS 1) cells. The hybridoma culture supernants were screened by indirect ELISA with artificial synthetic human inhibinαsubunit 37-65 fragment as antigen. Results: Three hybridoma cells lines which can secret McAbs were successfully obtained.The titer of ascites were 1×10 -5 ～1×10 -3 ,and the McAbs had no cross reaction with the INH α(1-32)and other polypeptide hormones.Epitope specificity analysis proved that 3 lines recogrinized the same epitope.Relative affinity of them were ⅠA5>Ⅳ3C>Ⅴ5G Antigen compitive inhibition test showed that the antibodies could be blocked by preincubating with human follicle fluid,porcine follicle fluid and INH α(37-65). Conclusion: According to the evidences of immunological and immunohistochemical identification, it is concluded that three hybridom cell lines have high sensitivity, high specificity and usefulness. These antibodies can be used in immunohistochemistry study of the normal and some tumor tissues in humans, as well as in animals such as pig and rat.