一种高通量免疫检测抗生素残留方法的建立

目的将表面具有羧基的聚苯乙烯微球运用于悬液芯片系统中,建立一种高通量检测牛奶中卡那霉素和氯霉素残留的方法。方法将合成的卡那霉素和氯霉素全抗原包被在聚苯乙烯微球表面构成捕获抗原,并对整个包被过程进行优化。利用捕获抗原、单克隆抗体及检测物构建间接竞争免疫检测体系,并用荧光标记的二抗作为荧光探针标记与捕获抗原结合的单克隆抗体得到悬液芯片系统的检测物。由于不同型号的微球被悬液芯片系统的激光激发后所呈现的侧向散射荧光(side scatter,SSC)和前向散射荧光(forward scatter,FSC)不同,所以悬液芯片系统能够将不同的微球进行区分以达到分辨不同检测物的目的,悬液芯片系统上的另外一套检测系统能够对每个微球表面荧光探针的荧光强度进行检测以实现定量分析,双检测系统同时工作实现了高通量检测的目的。结果微球的包被优化实验结果表明,包被100μL微球需要卡那霉素和氯霉素全抗原的最佳量分别是12μg和17μg。单克隆抗体的最佳稀释倍数分别是800倍和1600倍;在牛奶基质中,该方法对卡那霉素和氯霉素的检测限分别是0.79ng/mL和52.01ng/mL,半数抑制浓度(IC50)分别为36.65ng/mL和626.03ng/mL,检测范围分别为9~900ng/mL和90~900ng/mL;抗体特异性实验表明两种单克隆抗体的特异性良好。该方法在牛奶基质中的标准添加回收率为80%~110%,满足检测方法建立的要求。结论利用基于微球技术的悬液芯片系统建立了一种高通量检测牛奶中抗生素残留的方法,该研究为下一步更多种抗生素残留的同步检测提供了数据依据。

Establishment of a multiplexed immunoassay for residues of antibiotics

Objective A multiplexed microsphere-based immunoassay was developed for simultaneously detecting residues of kanamycin and chloramphenicol in raw milk by suspension array system.Methods Synthetic antigen of kanamycin and chloramphenicol were covalently coupled with fluorescent-coded microspheres to format a capture antigen.The indirect competitive reaction system was founded using monoclonal antibody,standard sample and capture antigen.Monoclonal antibodies were labeled by fluorescent probe as detector of suspension array system.The fluorescent of beads was excited by lasers of suspension array system.Fluorescent intensity from side scatter and forward scatter were different for different beads,so different beads,as different test substances,would appear different regions,which was very important for multiplexed test.The fluorescent intensity of fluorescent probe was detected by other detector of suspension array system to achieve quantitative analysis.Results The results showed that dosage of coupling beads with synthetic antigen of kanamycin and chloramphenicol were 12μg/100μLbeads and 17μg/100μLbeads,respectively.The best diluted multiple of monoclonal antibody against kanamycin and chloramphenicol were 800× and 1600×,respectively.The limit of detection for kanamycin and chloramphenicol in raw milk were 0.79ng/mL and 52.01ng/mL,respectively.Test scales of this immunoassay for kanamycin and chloramphenicol were 9~900ng/mL and 90~900ng/mL,respectively.Conclusion Beads-based suspension array system was used to detect residues of antibiotics in raw milk,which provided a date for advanced multiplexed detection on residues of antibiotics in another food.