抗伏马菌素B_1单克隆抗体的制备及鉴定

目的建立抗伏马菌素B1（FB1）的单克隆杂交瘤细胞株,制备抗FB1的单克隆抗体（McAb）。方法采用FB1-KLH偶联物小剂量长周期免疫BALB/c小鼠后,采用细胞融合方法获得分泌抗FB1的杂交瘤细胞株,采用多次亚克隆的方法建立了4株稳定分泌抗FB1抗体的杂交瘤细胞株。腹水诱生法获得大量McAb,采用抗体亚类测定试剂盒鉴定抗体的亚类,SDS-PAGE测定抗体分子量,McAb的特异性和灵敏度用间接竞争抑制ELISA。结果免疫小鼠血清检测结果显示经过5次免疫后血清的效价稳定在1×10-6,经过4次亚克隆后建立4株稳定分泌抗FB1抗体的杂交瘤细胞株,获得腹水,纯化抗体,抗体亚类属于IgG2a类,轻链为κ,轻链和重链的相对分子质量分别是55000和32000,ELISA检测抗体特异性显示可与FB1发生特异性反应,采用此抗体建立间接竞争抑制ELISA方法的线性范围在2～500ng/ml。结论制备了特异性和灵敏度较高的抗FB1单克隆抗体,为建立伏马菌素免疫学检测方法打下良好基础。

Development of monoclonal hybridoma cell lines against fummonisin B_1

Objective This study aims to prepare monoclonal antibody against fummonisin B 1（FB 1）.Methods FB 1-KLH and FB 1-BSA were used in the immunization of 6～8 weeks old female BALB /c mice.Immune spleen cells were fused with sp2 /0 myeloma cells to produce the hybridoma cell lines secreting McAb against FB 1.The cells were subcloned for the selection of stable antibody producing cell lines.McAbs was obtained from the ascites and then purified.Both the isotypes and the molecular weight were determined.The specificity and sensitivity of McAb were evaluated using the indirect competitive ELISA method.Results After 5 times of immunizations,the titer of serum antibody was 1×10-6.The McAb was found to be IgG2a subclass and the light chain was κ.The molecular weight of the heavy and light chain was 55 000 and 32 000,respectively.The reactivity of FB 1 was confirmed by the ELISA assay.The linear range of the antibody in the indirect competitive inhibition ELISA assay was 10～500 ng /ml.Conclusion The monoclonal antibody obtained for FB 1 is highly specific.