抗克伦特罗单克隆抗体的制备及鉴定

Preparation and Identification of the Monoclonal Antibody Against Clenbuterol

利用重氮化法,将小分子半抗原克伦特罗（CL）与牛血清白蛋白（BSA）偶联,制备得到CLBSA完全抗原,并用其免疫Balb/c小鼠,通过细胞融合、连续克隆筛选能稳定分泌高特异性抗CL的单克隆抗体细胞株。结果显示:使用偶联率为17的克伦特罗完全抗原免疫Balb/c鼠,间接ELISA法检测其中三只小鼠血清效价较高;细胞融合检测得到其融合率为85.26%,阳性率为15.23%;经四次连续克隆得到了一株特异性最佳的细胞株4H5,间接ELISA法检测其培养上清的OD450nm达到1.265±0.060,与BSA交叉反应OD450nm仅为0.060±0.006,腹水效价达3.2×106;最后经鉴定确定该单克隆抗体亚型为IgG1,轻链为κ。

The clenbuterol was conjugated with BSA by diazotization at a ratio of 17 to form CL-BSA complex.The conjugated molecule was injected into the Balb/c mice for hybridoma preparation.The results showed that the synthesized antigen could immune the mice successfully.The frequency of fusion and the positive cloning could reach 85.26%and 15.23%,respectively.Through four rounds of cloning,one optimal clone named 4H5 was obtained.The result of IELISA demonstrated that the 4H5 culture supemament was high specific activitie against CL and could reach to 1.265 ± 0.060(OD450nm),comparing to the ascites against BSA(OD450nm 0.060 ±0.006).The titer of ascites generated from 4H5 injection could reach to 3.2×10~6 with low affinity toward BSA,and the subtype is IgG1 with light chain k.