恩诺沙星单克隆抗体的制备及鉴定

 碳二亚胺法合成了2种恩诺沙星(enrofloxacin,ENFX)人工抗原ENFX-BSA和ENFX-OVA,用紫外扫描和分析载体蛋白的氨基酸组成等方法对人工抗原进行鉴定,ENFX与BSA的结合比约为48∶1。用ENFX-BSA免疫BALB/C小鼠,采用显微克隆技术最终筛选出2株特异性分泌细胞2C5和5D5并获取腹水,纯化后腹水效价分别为1∶3200和1∶6400。2C5和5D5均属IgG2a型抗体。单抗5D5间接竞争ELISA(Ci-ELISA)的工作浓度为1∶1000,ENFX的抑制曲线在0.13～10

Studies on Preparation and Characterization of Monoclonal Antibodies Against Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

An ester activation method was employed to synthesize enrofloxacin (ENFX) carring protein BSA andOVA. The conjugates ENFX-BSA and ENFX-OVA were identified by UV and amino acids automation analysis instrument,and resulted in conjugates with 48 ENFX molecules per carrier molecule (BSA), Splenocytes from mice immunized withENFX-BSA were fused with SP2/0 myeloma cells, and hybridomas secreting antibodies against enrofloxacin were selectedand cloned. Two stable monoclonal antibodies, 2C5, 5D5 of subclasses IgG2a, were isolated, Using 5D5, an indirect competitiveinhibition enzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detection of enrofloxacinand its metabolites. The IC50 of standard curve was 21.67ngml-1 and the limit of detection for enrofloxacin was 0.13 ngml-1.This method was sensitive and had a linear range from 0.13 to 10 000 ngml-1 (r = -0.9782). Monoclonal antibodies 5D5exhibited high relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin, marbofloxacin, sarafloxacin anddanorfloxacin were 110.8%, 27.40%, 71.05% and 37.41%, respectively. Three non-fluoroquinolones of cefadroxil,chloramphenicol, and sulfamonomethoxine were tested and there was no cross-reaction between them.