人抑制素βB亚基单克隆抗体的制备和鉴定

Preparation and identification of human inhibin β~B subunit monoclonal antibody

目的：人抑制素βＢ亚基单克隆抗体（单抗）的制备和鉴定。方法：以化学合成人抑制素βＢ（１－ ２８） 和（８５－１１５）多肽片段和粗提猪卵泡抑制素免疫ＢＡＬＢ／ｃ 小鼠，经细胞融合与筛选，得到分泌单抗的细胞株，诱生腹水后采用特定的免疫学方法进行鉴定。结果：获得４ 株能分泌单抗的细胞株，ＥＬＩＳＡ 检测腹水效价为１ ×１０－４ ～１ ×１０－ ６，与α、βＡ亚基片段及其他多肽激素无交叉反应，３ 株ＭｃＡｂ（ 抗ＩＮＨβＢ（８５－ １１５）） 识别同一抗原位点。相对亲和力３Ｅ８＞５Ｅ１１＞７Ｂ９。腹水中的抗体能明显地被人卵泡液，猪卵泡液和βＢ（８５ －１１５）ＢＳＡ 中和而呈抑制曲线。

Aims: To prepare the McAb againsting the INH βB subunits and to identify them. Methods:BALB/c mice were immunized with crude preparation of porcine inhibin by intra peritoneal injection,and followed by injection with chemically synthetic peptide INHβB (1-28) and βB(85-115) directly into the spleen of the immunized BALB/c mice 3 days before fusion. After fusion,selection and cloning producers,we got the cell lines which can secret McAbs,and used them to induce the ascites, then identified the McAb using specific immunological methods. Results:4 hybridoma cell lines which can secret McAbs were successfully obstained. The titer of ascites were 1×10\+\{-4\}～1×10\+\{-6\}, and the McAbs had no cross reaction with the α and βA subunits and other polypeptide hormones. Epitope specificity analysis proved that three cell lines (against INHβΒ(85-114)) recognized the same epitope. Relative affinity of them were 3E8>5E11>7B9. Antigen competitive inhibition test showed that the antibodies could be blocked by preincubating with human follicle fluid, porcine follicule fluid and INHβB(85-115) BSA. Conclusion: We first estabolished the cell lines which can secret the INH βB subunit's antibodies having high specificity and high affinity in China.