诺氟沙星单克隆抗体的制备及其特性鉴定

Development of monoclonal antibody against Norfloxacin

为制备诺氟沙星单克隆抗体(MAb),用碳二亚胺法将诺氟沙星(Norfloxacin,NFLX)分别偶联于载体蛋白牛血清白蛋白(BSA)和卵清蛋白(OVA),合成免疫原BSA-NFLX和包被原OVA-NFLX,并用紫外(UV)扫描、SDS-PAGE电泳和免疫小鼠进行鉴定;用BSA-NFLX免疫BALB/C小鼠,间接ELISA和阻断ELISA选择细胞融合备用鼠;应用杂交瘤技术建立分泌NFLX MAb的细胞株,用体内诱生腹水法制备NFLX MAb;对NFLX MAb的效价、敏感性和特异性等特性进行测定。结果表明:NFLX人工抗原制备成功,融合筛选后获得3C6、3G11两株特异敏感的杂交瘤细胞,两株细胞培养上清液效价为1∶512,腹水效价为1∶6.4×105,3C6株对NFLX的IC50为2.52μg/L,与二氟沙星、沙拉沙星有小于0.03%的交叉反应性,与其他抑制物无交叉反应性。本实验获得了高效价、敏感、特异的抗NFLXMAb,为NFLX残留快速检测方法的建立奠定了基础。

Monoclonal antibody （MAb） directed against Norfloxacin was developed by fusion of myeloma cells with spleen cells obtained from mice immunized with BSA coupled norfloxacin antigen （BSA-NFLX）. Two hybridoma cell lines,3C6 and 3G11 that secret MAb specific for NFLX were established. The titer of the MAb determined by indirect ELISA were 1 ∶512 in supernatant and 1∶6.4×105 in ascites,repectively. The MAb secreted by 3C6 showed an IC50 of 2.52 μg/L and had less than 0.03% cross-reactivity with difloxain and sarafloxacin. This MAb has a high titer and is specific and highly sensitive which could be used for immunoassays to detect NFLX residues.