二氟沙星单克隆抗体的研制及其免疫学特性的鉴定

Trituration and Immunologic Traits of Monoclonal Antibody for Difloxacin

采用改进的碳二亚胺法,将半抗原二氟沙星(DIF)与载体牛血清白蛋白(BSA)和卵清白蛋白(O-VA)偶联,制备得到二氟沙星全抗原DIF-BSA和DIF-OVA。采用紫外(UV),凝胶电泳(SDS-PAGE)进行鉴定;用BSA-DIF免疫BALB/C小鼠,间接ELISA和阻断ELISA选择细胞融合备用鼠;应用杂交瘤技术制备二氟沙星单克隆抗体(DIF mAb),并对其效价、亲和力和特异性等进行鉴定。结果表明,BSA与DIF偶联成功,分子结合比为1:4.7,筛选出1H10、2F12、4D8共3株敏感特异的杂交瘤细胞,间接ELISA效价细胞培养上清分别为1:5.12×102、1:3.2×101、1:2.56×102,腹水效价分别为1:2.56×105、1:1.28×105、1:6.4×104。同种亚型分别为IgG1、IgG1、IgG2a,亲和常数(Ka)分别为2.94×1010L/mol、1.72×1010L/mol和1.35×1010L/mol。亲和力最高的1H10对DIF的IC50为2.2ng/ml,单抗与其他氟喹诺酮类药物和磺胺类药物无交叉反应。通过试验获得高效价、敏感、特异的mAb,适用于动物性食品中DIF的残留检测的免疫学试验。

Difloxacin （DIF） was coupled with carrier protein bovine serum albumin （BSA） and ovalbumin （OVA） to prepare complete antigen DIF-BSA and DIF-OVA by modified method of carbodiimide. Both UV scanning spectrum and SDS-PAGE were used to identify DIF artificial antigen. BALB/C mice were immunized with BSA-DIF, the titre of monoclonal antibody （mAb） was directed by indirect ELISA and blocking ELISA. The hybridoma lines that secrete DIF mAb were established with using hybridoma technology. The immunological traits such as titer, affinity and specificity of the mAb were characterized. The results showed that the complete antigens DIF-BSA was successfully coupled and the conjugation ratio of DIF to BSA was about 4.7：1. There hybridoma cell lines of 1H10, 2F12 and 4D8 were screened for specificity to DIF, the indirect ELISA titer of the mAb were 1：5.12×10^2, 1：3.2×10^1, 1：2.56×10^2 in supernatant, 1：2.56×10^5, 1：1.28×10^5, 1：6.4×10^4 in ascites, the isotypes of the mAb were IgG1, IgG1, IgG2a,and the affinity constant （Ka） were 2.94×10^10L/mol, 1.72×10^10L/mol and 1.35×10^10L/mol, respectively, the mAb of 1H10 showed good sensitivity with IC50 of 1.64ng/ml to DIF. mAb has no cress-reactivity to other FQS and sulfonamides. DIF mAb of high-titer, sensitivity and specificity had been generated, it is possible to establish immunoassay of DIF residues in animal food.