氟罗沙星残留检测间接竞争ELISA试剂盒的研制

doi:10.3864/j.issn.0578-1752.2014.11.019

摘要/Abstract

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30 μg/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：5 μg•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（R2 = 0.9956），与同系药物及其它药物均无明显交叉反应，阳性猪肝样的回收率在67.5%—87.9%，平均78.7%，变异系数在9.34%—10.7%，平均10.0%；阳性猪肉样的回收率在74.0%—88.2%，平均81.42%，变异系数在8.3%—10.4%，平均9.48%；平均变异系数均小于15%;在500、250、150、75、30 ng•mL-1 5个标准浓度 B/B0的批内变异系数在3.39%—6.68% ，批间变异系数在4.69%—9.67%。批内和批间平均变异系数分别为5.07%和7.44%，且与HPLC方法具有良好的一致性(R2=0.9988)，通过实际测定其结果基本符合2010年版《中国药典》中所规定的含量要求(90%—110%)，进一步说明二者具有良好的相关性以及FLE-Kit的准确性。【结论】首次利用两种方法分别合成FLE免疫原和包被原，利用所制备的抗FLE血清建立并优化FLE残留icELISA检测方法，制备出FLE快速检测试剂盒。该试剂盒具有灵敏、准确、简便、快速的特点，为氟罗沙星免疫学快速检测方法的建立奠定了坚实的基础。

关键词: 氟罗沙星 , 人工抗原 , 多抗血清 , ELISA , 试剂盒

Abstract: 【Objective】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 189 μL of conjugate in 311 μL 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 189 μL of FLE-BSA in 311 μL 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 2.5×104 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%. Good agreement of the results obtained by ELISA and high performance liquid chromatography (HPLC) further confirmed the reliability and accuracy of the icELISA for rapid detection of SPFX in pork meats. The coefficients of variation of intra-assay and inter-assay were 5.07% and 7.44% during five standard concentrations. The antiserum of FLE had no cross reactivity with other competitors.【Conclusion】The FLE polyclonal antiserum has been generated. The icELISA method and rapid detection kit were developed for the detection of FLE residues with the characters of sensitivity, accuracy, convenience and rapid in this study and they laid a foundation for establishing immunoassay of FLE residues.

Key words: fleroxacin , artificial antigen , polyclonal antiserum , ELISA , kit

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. 氟罗沙星残留检测间接竞争ELISA试剂盒的研制[J]. 中国农业科学, 2014, 47(11): 2251-2261.

JIA Guo-Chao-1, 2 , ZHI Ai-Min-1, LI Meng-Qin-2, SONG Chun-Mei-1, WANG Ling-Ling-1, LIU Ru-Biao-1, HU Xiao-Fei-1, WANG Fang-Yu-1, ZHANG Gai-Ping-1. Study on Rapid Detection Kit of Fleroxacin by icELISA[J]. Scientia Agricultura Sinica, 2014, 47(11): 2251-2261.

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