抗人ATⅢ单克隆抗体的研制

Preparation of Monoclonal Antibodies against Human Antithrombin Ⅲ

目的：建立人抗凝血酶Ⅲ（ Ａ Ｔ Ⅲ）的 Ｅ Ｌ Ｉ Ｓ Ａ 检测试剂盒及 Ａ ＴⅢ的纯化提供试剂准备。方法：用 Ａ ＴⅢ进行 ２ 次基础免疫、２ 次加强免疫的方法免疫 Ｂａｌｂ／ｃ 小鼠，经间接 Ｅ Ｌ Ｉ Ｓ Ａ 的方法筛选杂交瘤细胞株，通过吸收试验、免疫印迹试验等进行特异性分析。结果：筛选出１５ 株分泌抗人 Ａ ＴⅢ单克隆抗体的杂交瘤细胞株，对其中３ 株（ Ｂ４、 Ｂ５、 Ｂ７）作进一步研究，３ 株杂交瘤细胞分泌抗体亚型均为 Ｉｇ Ｇ１ 型，３ 株细胞培养上清滴度平均达到４×１０３，小鼠腹水滴度平均达到 ４×１０６，３ 株抗体均具抗人 Ａ ＴⅢ的特异性。结论：所筛选的单抗为 Ｅ Ｌ Ｉ Ｓ Ａ 检测试剂盒的制备及 Ａ ＴⅢ的纯化提供了试剂准备。

Objective:To develop ELISA reagent kit for detection of human antithrombin Ⅲ(ATⅢ) and to make reagent preparations for purification of ATⅢ.Methods:The immunifaction of Balb/c mice was performed by twice basic immunization and twice booster immunization with ATⅢ.The hybridoma strains were screened by indirect ELISA method.The spectificity analysis was carried out by competitive absorbency test,Western blotting test and so on.Results:15 hybridoma strains which secreted monoclonal antibodies against human ATⅢ,were screened out,and then the further research on 3 strains of them:B4,B5 and B7 was conducted.The subclasses of monoclonal antibodies secreted by those 3 strains were IgG 1.The average titer of culture supernantant of hybridoma of those 3 strains reached 4×10 3.The average titer of ascitic fluids of mice reached 4×10 6.The antibodies secreted by those 3 strains were specific against human ATⅢ.Conclusion:The screened monoclonal antibodies could make the ragent preparations for production of ELISA reagent kit used in the detection and purification of ATⅢ.