氟罗沙星残留检测间接竞争ELISA试剂盒的研制  

Study on Rapid Detection Kit of Fleroxacin by icELISA

作　　者：贾国超[1,2] 职爱民[1] 李梦琴[2] 宋春美[1] 王玲玲[1] 刘儒彪[1] 胡骁飞[1] 王方雨[1] 张改平[1] 

机构地区：[1]河南省农业科学院/农业部动物免疫学重点实验室/河南省动物免疫学重点实验室,郑州450002 [2]河南农业大学食品科学技术学院,郑州450002

出　　处：《中国农业科学》2014年第11期2251-2261,共11页Scientia Agricultura Sinica

基　　金：国家科技支撑计划(2011BAK10B01);农业部公益性行业(农业)科研专项(201003008-09)

摘　　要：【目的】利用蛋白质连接技术合成氟罗沙星(FLE)人工抗原,制备FLE高亲和力抗体,通过建立、优化FLE的icELISA检测方法研制出快速、灵敏的FLE残留检测间接竞争ELISA试剂盒。【方法】用DCC法偶联FLE和载体蛋白BSA合成免疫原FLE-BSA,用混合酸酐法偶联FLE和载体蛋白OVA合成包被原FIE-OVA,通过紫外扫描法(UV Scan)和聚丙烯凝胶电泳法(SDS-PAGE)鉴定人工合成抗原。选择经初步鉴定成功合成的FLE-BSA免疫SPF级6—7周龄Balb/c雌性小鼠5只,采用多点免疫法,对小鼠背部皮下4-6点注射免疫,免疫剂量为30μg/只,初免,用FLE-BSA PBS溶液与等体积FCA混合乳化后免疫;20 d后用FLE-BSA PBS溶液与等体积FIA混合乳化后免疫,免疫间隔3周,4次免疫后,利用间接ELISA和间接竞争ELISA测定其多抗血清效价和敏感性,选出效价高、敏感性好的小鼠多克隆血清,建立、优化FLE的icELISA检测方法,确定其包被浓度、包被时间、封闭液选择、封闭时间、抗体工作浓度、二抗稀释度、底物显色时间等条件,制备出FLE快速检测试剂盒,同时测定该试剂盒的灵敏度、精密度、准确度以及特异性等指标,并与高效液相色谱法做相关性比较,同时对市场上购买4个批次的氟罗沙星药品含量进行了实际测定,以确证试剂盒在实际应用中的质量。【结果】通过上述蛋白质连接技术所制备的免疫原经紫外扫描法和聚丙烯凝胶电泳法鉴定,结果显示BSA与FLE偶联后,FLE-BSA波峰整体出现右移且凝胶上BSA的泳动速度大于FLE-BSA,初步证明人工合成抗原偶联成功。通过间接ELISA和间接竞争ELISA方法测定5号小鼠多抗血清得到其效价大于2.5×104,抑制价为162.18 ng·mL-1。通过优化FLE icELISA检测方法,其工作条件:包被浓度:5μg·mL-1;抗体工作浓度:1﹕6400;二抗稀释度:1﹕1000;底物显色时间:10 min。所制备的试剂盒灵敏度为16.22 ng·mL-1,标准曲线回归方程为y=-0.3627x+1.3517(R2Objective The goals of this study were to obtain immunogen and coating antigen, generate its mice polyclonal antiserum and develop FLE-Kit by its competitive indirect enzyme-linked immunosorbent assay （ciELISA）.[Method]Artificial antigen BSA-FLE and OVA-FLE were synthesized using DCC and the mixed anhydride reaction methods by linking carrier proteins BSA and OVA to FLE. The antigens BSA-FLE and OVA-FLE were identified by ultraviolet scanning and SDS-PAGE, then five female white rats were subcutaneously immunized with the immunogen Fle-BSA at multiple sites in the back. The initial immunization was subcutaneously injected with 189μL of conjugate in 311μL of PBS （0.01 mol·L-1, pH 7.4） and 0.5 mL of Freund’s complete adjuvant. The rest of five booster immunizations were conducted by injecting 189μL of FLE-BSA in 311μL of PBS （0.01 mol·L-1, pH 7.4） and 0.5 mL of iFA at 20-day intervals. After obtained mice polyclonal antiserum, the ciELISA method and rapid detection kit of FLE were developed. The kit was compared with HPLC method to ensure its quality.[Result]The ultraviolet scanning and SDS-PAGE showed that FLE artificial antigen was synthesized successfully. Five BALB/c mice indirect ELISA titer against FLE were all above 2.5×10^4 and the IC50 of No.4 mice was the lowest （162.18 ng·mL^-1）. After optimized the ELISA method, the icELISA revealed that the optimal concentration of mice serum was 1：6400, the optimal concentration of coated antigen was 5 μg·mL^-1, and the optimal concentration of sheep anti-rabbit IgG was 1：1000. The regression equation was Y=-0.3627x＋1.3517（R2=0.9956）, the lowest detection limit of the kit was 16.22 ng·mL^-1, the assay measured drug residue in pork liver spiked with FLE with coefficient of variation between 9.34%-10.7%, and the average recovery rates between 67.5%-87.9%, respectively. The assay measured drug residue in pork meats spiked with FLE with coefficient of variation between 8.3%-10.4%, the average recovery rates were between 74%-88.2%. Go

关 键 词：氟罗沙星 人工抗原 多抗血清 ELISA 试剂盒 

分 类 号：S859.84[农业科学—临床兽医学]