抗鸡蛋清提取物单克隆抗体的制备与鉴定

Preparation and Characterization of Monoclonal Antibodies Against Purified Egg White Extract

目的:制备和鉴定小鼠抗鸡蛋清提取物单克隆抗体。方法:以纯化后的蛋清提取物为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选可特异性分泌抗蛋清提取物McAb的杂交瘤细胞。采用Ig类与亚类鉴定试剂盒鉴定McAb的Ig类与亚类;通过间接ELISA、Western Blotting鉴定McAb的特异性。结果:获得2株可稳定分泌抗蛋清提取物McAb的的杂交瘤细胞,分别命名为5E12和4F1,其分泌的McAb均为IgG1。Western Blotting分析表明,McAb 4F1所识别的靶分子的相对分子质量(Mr)约为44kD。间接ELISA法检测两株McAb5E12、4F1的冻融前后效价分别为1:3000、1:300。结论:成功制备了两株抗蛋清提取物McAb、5E12和4F1,为变应原的提取、纯化及鉴定提供了有效的实验基础。

Objective： To prepare and identify monoclonal antibodies （McAbs） against purified egg white extract. Methods： BALB/c mice were immunized with purified extracts of eggs, and hybridoma cell lines were obtained by fusing immunized mouse spleen cells with myeloma NS-1 cells. The specificities of McAbs were characterized by indirect ELISA and Western Blotting. Results： Two hybridoma cells, 5E12 and 4F1, which secreted the McAbs （both are IgG1） against extracts of eggs, are obtained. The titers of 5E12 and 4F1 are 1：3000 and 1：30, respectively. Western Blotting analysis showed that 4F1 can recognize a target molecule with relative molecular mass of 45 kD. The tesults of indirect ELISA showed that the titers of two McAbs are 1：3000 and 1：300, respectively. Conclusion： Two McAbs, 5E12 and 4F1, against extracts of eggs are prepared successfully, which provides valuable experiment foundation for quickly measuring allergen in foods as well as for isolating, purifying and identifying allergens.