氟罗沙星单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody Against Fleroxacin

采用碳二亚胺(EDC)法将氟罗沙星(FLE)与载体蛋白BSA、OVA分别偶联合成人工抗原BSA-FLE、OVA-FLE,通过聚丙烯酰胺凝胶电泳法和紫外分析法对合成的人工抗原进行了鉴定。BSA-FLE用作免疫抗原免疫Balb/C小鼠,OVA-FLE用作检测抗原包被酶标板检测抗体效价。免疫小鼠的脾细胞与SP2/0细胞融合获得了5株(4F11、2C3、8G2、3C1、2G8)稳定分泌抗FLE抗体的杂交瘤细胞株,抗体亚型鉴定均为IgG1。将3C1接种小鼠制备腹水,用辛酸-硫酸铵法对腹水进行纯化,纯化后腹水效价1∶1 024 000。抗体分子量160.766 KD,亲和常数为8.57×108M-1。

Fleroxacin was conjugated directly to BSA and OVA respectively by EDC method.The BSA-FLE was used as immune antigen to inoculate Balb/C mice and the OVA-FLE was used to coat the ELISA plate to detect the antibody titer.Splenocytes from mice immunized with BSA-FLE were fused with SP2/0 myeloma cells.Five hybridomas of stable secreting FLE-McAb were selected and were named 4F11,2C3,8G2,3C1 and 2G8.The subclass of all these five McAbs was IgG1.3C1 was inoculated to mice to prepare ascites.Ascites was purified by caprylic acid/ammonium sulfate precipitation（CA-AS） method.The titer of the purified ascites was 1∶1 024 000.Molecular weight of the McAb was 160.766 KD,affinity constant was 8.57×108 M-1.