摘要
目的:建立一种检测氯霉素(chloramphenicol,CAP)和克伦特罗(clenbuterol,CL)兽药残留的新型高通量悬浮芯片技术。方法:合成2种兽药的牛血清白蛋白(bovine serum albumin,BSA)结合物,并进行紫外和质谱鉴定。将2种兽药的BSA蛋白结合物偶联于悬浮芯片的固相载体——聚苯乙烯荧光微球上,在液相反应体系中,2种小分子兽药抗原和微球上的结合物共同竞争液相中各自特异性的生物素化单抗,优化和筛选出微球上偶联BSA结合物和反应抗体的最适加入量。绘制2种兽药残留检测的标准曲线,进行特异性检测和盲样的测定。结果:2种小分子兽药可与BSA成功偶联,CAP和CL与BSA的偶联比分别为40∶1和31∶1。经过优化,2种结合物的最适加入量均为5μg/100μl羧基微球;最佳抗体加入量分别为5和1ng/2000个反应微球。检测的标准曲线方程和方程相应的决定系数分别为:YCAP=-250.323+4103.517/[1+(X/30121.243)0.980],r2=0.994和YCL=-263012.682+265751.765/[1+(X/6.063)0.115],r2=0.989。2种兽药悬浮芯片的检测区间分别为40～6.25×105ng/L和50～7.81×105ng/L;最低检出限为:40ng/L和50ng/L。CAP和CL最低检出限均低于国标。同时,悬浮芯片的特异度测试良好,与其他药物无明显交叉反应。悬浮芯片对盲样测定的检测浓度值与实际浓度偏差较小。扫描电镜对微球表面微观结构的观察也直观地确证了蛋白在微球上的成功偶联。结论:高通量悬浮芯片技术操作简单,灵敏快速,成本低廉,为多种兽药残留的快速检测提供了新方法,具有广阔的应用和发展前景。
Objective：To establish a novel suspension array technology for the detection of chloramphenicol（CAP） and clenbuterol（CL） residues. Methods： Two conjugates coupling the 2 veterinary drugs with BSA were synthesized and identified by UV spectrophotometry and mass spectrometry. Then, they were immobilized on the solid carrier of the suspension array--the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted, and the specificity and the unknown samples were also determined. Results： Successful coupling was completed between small molecular veterinary drugs and BSA. The coupling ratio of CAP and CL with BSA was 40：1 and 31：1 ,respectively. The optimum amounts of the two conjugates were 5 μg per 100 μl beads and that of the two antibodies were 5 and lng per 2000 beads,respectively. The detective standard curves and their corresponding coefficients of determination were YCAP= -250.323 ＋4103. 517/[ 1 ＋ （X/30121.243）^0.980 ], r^2 = 0. 994 and YCL = - 263012. 682 ＋ 265751. 765/[ 1 ＋ （X/ 6. 063 ）^0.115], r^2 = 0.989. The detection ranges of the two veterinary drugs were 40 -6.25×10^5 ng/L and 50 -7.81×10^5 ng/L, respectively. The lowest detection limits of CAP and CL were below National Standards（GBs）. Simultaneously, the specific detection of the suspension array was excellent and did not indicate significant cross-reactions with other drugs. Errors between the detected and the real concentration for the detection of the unknown samples were relatively small. Conclusion： The high-throughput suspension array provides a novel method for analysis of multiple veterinary drugs and has a good application, being user-friendly, highly sensitive, high-speed and cost-effective.
出处
《军事医学科学院院刊》
CSCD
北大核心
2009年第1期41-44,65,共5页
Bulletin of the Academy of Military Medical Sciences
基金
国家科技支撑计划(2006BAK02A09)
国家自然科学基金资助项目(30771810)
关键词
氯霉素
克伦特罗
药物残留
悬浮芯片
微球
中位荧光强度
chloramphenicol
clenbuterol
drug residues
suspension array
microspheres/beads
median fluorescent intensity （MFI）
