喹乙醇单克隆抗体的制备及其免疫学特性的鉴定

PREPARATION AND IMMUNOLOGIC TRAITS OF MONOCLONAL ANTIBODY FOR OLAQUINDOX

通过质谱法鉴定合成喹乙醇-半琥珀酸酯(OLA-HS),紫外法及凝胶电泳法鉴定全抗原OLA-BSA和OLA-OVA后,使用杂交瘤技术制备喹乙醇单克隆抗体(OLA mAb),对其效价、亲和力、敏感性、特异性及亚型进行鉴定,并通过体内诱生腹水法进行大量制备。试验结果表明,本研究成功制备了OLA-HS,且半抗原与载体蛋白偶联成功;筛选出的5株杂交瘤细胞单抗同种型均为IgG1,细胞培养上清间接ELISA效价在1∶3.0×102～1∶1.28×103之间,用4E12细胞株制作的腹水效价为1∶5.12×105。OLAmAb亲和常数Ka为3.75×1010L/mol,对OLA的IC50为1.51ng/ml,且具有较好的特异性。试验获得了高效价、敏感、特异的OLA mAb,可用于动物性食品中OLA残留检测的免疫学试验。

Anti-olaquindox monoclonal antibody（OLA mAb）,was prepared by hybridoma technology,and the titer,affinity,sensitivity,specificity and isotype of mAb were analyzed.The OLA derivative,OLA-HS was synthesized by succinic anhydride method and identified by ESI-MS.Two complete antigens of OLA-BSA and OLA-OVA were characterized by methods of ultraviolet spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis,respectively.OLA mAb ascites were induced using an in vivo method for mAb production.The results showed that OLA-HS had been synthesized,and the OLA-HS had been conjugated to the carrier protein.Five hybridoma cell lines were screened,and all the isotypes of the mAb were IgG1.The indirect ELISA titer of the mAbs were 1∶ 3.0 × 102 ～ 1∶ 1.28 × 103 in supernatant,and the titer of hybridoma cell line 4E12 was 1∶ 5.12 × 105 in ascites.The affinity constant（Ka） was 3.75 × 1010 L /mol,the special mAb of 4E12 showed good sensitivity with IC50 of 1.51ng /ml to OLA.OLA mAb with high-titer,sensitivity and specificity had been produced successfully,which provide essential reagents to develop the immunoassay for OLA residues in food of animal origin.