恩诺沙星抗体免疫检测条件的建立

Studies on immunodetection of antibodies against enrofloxacin

目的制备恩诺沙星(ERLX)的多克隆抗体,以期建立通过酶联免疫吸附试验(ELISA)检测恩诺沙星抗体的效价和优化快速检测方法。方法将牛血清白蛋白(BSA)和鸡血清白蛋白(OVA)偶联成功的恩诺沙星完全抗原(BSA-ERLX和OVA-ERLX)免疫新西兰家兔得到的血清分别保存在4、-20和-70℃半年后,用ELISA方法确定抗原包被的最适浓度、血清(一抗)的效价、一抗的最适稀释倍数及二抗的最适稀释倍数。首先将抗原包被浓度分为三组:20、10和5μg/ml,确定最适包被浓度,并在此基础上确定血清的效价和血清的最适稀释倍数。结果不同温度下保存的BSA-ERLX和OVA-ERLX抗体的最适抗原包被浓度均为10μg/ml。在4、-20和-70℃保存的BSA-ERLX的抗体效价分别为12800、25600和25600;一抗的最适稀释倍数分别是:800、800和1600,二抗最适稀释倍数为1000。OVA-ERLX的效价在4、-20和-70℃分别为3200、6400和6400;一抗的最适稀释倍数分别为800、1600、1600,二抗的最适稀释倍数均为1000。结论不同温度下保存的BSA-ERLX比OVA-ERLX抗体效价均高,综合考虑认为,-20℃保存的BSA-ERLX抗体,更加适合于进行ELISA检测。

Objective To prepare the polyclonal antibodies of enrofloxacin （ERLX）, and examine the titer of antibody by the enzyme linked immunosorbent assay （ELISA） , in order to establish the optimized and fast examination method. Methods Bovine serum albumin （BSA） and ovalbumin （OVA） coupling enrofloxacin complete antigen （BSA-ERLX and OVA-ERLX） were used to immunize New Zealand domestic rabbits, serum obtained from the immuned rabbit was stored at 4℃, - 20℃ and - 70℃, respectively. After half year, the optimal concentration of coating antigen, the titer of antiserum, the suitable response concentration of antibody and the second antibody＇s optimal dilution multiples were determined with the ELISA method. The coating antigens were divided into three groups： 20μg/ml, 10μg/ml and 5μg/ml firstly, the most suitable coating concentration was determined, then the titer and the most suitable dilution multiple of the antiserum were tested. Results Though BSA-ERLX and OVA-ERLX antibody preserved under different temperatures, the most suitable coated antigen concentrations were both 10μg/ml. The titer of BSA-ERLX antibody preserved at 4℃, - 20℃ and - 70℃ were 12, 800, 25, 600 and 25, 600 respectively; and the most suitable dilution multiple of antibody were 800, 800 and 1,600 respectively, the optimal dilution multiples of second antibody was 1000. The titer of OVA-ERLX antibody preserved at 4℃, -20℃ and -70℃ were 3, 200, 6, 400 and 6, 400 respectively; and the suitable dilution multiple of antibody were 800, 1,600 and 1,600 respectively, the optimal dilution multiples of second antibody was 1000. Conclusion The titer of BSA-ERLX antibody stored at the different temperatures was higher than that of the OVA-ERLX antibody, and the antibody of BSA-ERLX stored at - 20℃ was more suitabled for the ELISA examination if considering all factors.