恩诺沙星快速酶联免疫吸附法检测试剂盒的研制

Development of Enrofloxacin ELISA Test Kit

目的:研制恩诺沙星酶联免疫吸附法检测试剂盒。方法:采用EDC-NHS法制备ENR-M-BSA免疫原和ENR-OVA检测抗原,用ENR-M-BSA免疫BALB/C小鼠,采用甲基纤维素半固体培养基法筛选单克隆抗体并采用间接竞争ELISA对其进行分析。结果:通过公式计算ENR-M-BSA摩尔偶联比为15:1,ENR-OVA摩尔偶联比为7.2:1。筛选出1株特异性分泌株3E9,其腹水纯化后间接竞争ELISA测其效价为107,分型试剂盒测试显示此抗体属于IgG1型;检测限为1ng/mL,线性范围在1～100ng/mL之间,半量抑制浓度(IC50值)为10ng/mL,与4种结构类似物交叉反应均小于1%。结论:此株单克隆抗体可以用来建立恩诺沙星ELISA检测试剂盒。

An enzyme-linked immunoabsorbence（ELISA） test kit was developed for enrofloxacin（ENR） detection.ENR-M-BSA and ENR-OVA antigens were prepared by EDC-NHS method.BALB/C mice were immunized with ENR-M-BSA for monoclonal antibody generation.A specific monoclonal antibody was screened using methylcellulose-based semi-solid medium and analyzed using indirect competition ELISA.The molar coupling ratios of ENR-conjugated BSA and ENR-conjugated OVA were calculated to be 15：1 and 7.2：1,respectively.The specific monoclonal antibody 3E9 was obtained and the titer of purified 3E9 antibody reached 107 according to ELISA detection,and belonged to the family of IgG1 according to the detection using an antibody typing kit.The ELISA detection of ENR using the antibody exhibited a sensitivity of 1 ng/mL,a linear range of 1 to 100 ng/mL and a half inhibitory concentration（IC50） of 10 ng/mL.The cross-reactivity between the ELISA detection kit developed and four analogues was less than 1%.These results indicate that the monoclonal antibody has a promising potential in the development of ELISA test kits for ENR detection.