二氟沙星残留检测用单克隆抗体的制备及其鉴定

用DFX-BSA免疫BALB/c小鼠,采用显微克隆技术成功筛选出5株特异性分泌细胞株1a6、2b4、4a4、X1和X2。将X1株细胞株直接注入小鼠腹腔获取腹水,腹水纯化后效价达1:1638400。蛋白质含量为6.86mg/ml,单抗X1属IgG2a亚型。X1株单克隆抗体的检测标准曲线方程为Y=0.4813-3.1067X(R=0.9510),最低检测限(LOD)为2ng/ml,线性范围为0.002-5μg/ml,DFX对其IC50为0.085μg/ml。单抗对氟喹诺酮类药物有一定的交叉反应,对沙拉沙星最高可达10.9%,对兽医临床常用抗菌药物氨苄西林、卡那霉素、盐酸四环素、磺胺甲噁唑和氯霉素等没有交叉反应。Ci-ELISA反应中McAb对低浓度的乙腈和丙酮具有良好的耐受性,而低浓度的甲醇对McAb的结合力有一定的影响。

Studies on Preparation and Characterization of Monoclonal Antibodies Against Difloxacin

Splenocytes from mice immunized with DFX-BSA were fused with SP2/0 myeloma cells, and hybridomas secreting antibodies against difloxacin were selected and cloned. Five stable monoclonal antibodies named 1a6, 2b4, 4a4, X1, X2 were isolated. An indirect competitive inhibition enzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detection of difloxacin. The IC50 of standard curve was 0.085μg/ml and the limit of detection for difloxacin was 2μg/ml. The method was sensitive and had a linear range from 0.002 to 5μg/ml (R=0.9510). Monoclonal antibodies exhibited relative affinity to sarafloxacin, with a cross-reactivity of 10.9%. Ampicillin, Kanamycin, Tetracycline, Sulfamethoxazole and chloramphenicol were tested and no cross-reactions were observed.