摘要
该研究建立了一种基于吖啶酯-磁微球的自动化学发光免疫法(Chemiluminescence Immunoassay,CLIA)检测牛奶中的赭曲霉毒素A(Ochratoxin A,OTA)。采用磁微球偶联二抗,吖啶酯标记OTA-BSA偶联物,优化了磁微球浓度、OTA抗体浓度、吖啶酯标记物浓度、抗体和标记物稀释液、反应时间、样品预处理条件等各种CLIA方法的试验条件。结果表明,CLIA方法对OTA的检出限为6.38 ng/kg,半抑制浓度(IC_(50))为31.40 ng/kg,线性范围为11.23~99.20 ng/kg,批内和批间的变异系数<8%;测定牛奶样品中的OTA时,检出限为25.52 ng/kg,IC_(50)为125.60 ng/kg,线性范围为44.92~396.80 ng/kg,添加回收率为95.08%~105.83%;对赭曲霉毒素B、赭曲霉毒素C的交叉反应率为6.52%和24.57%,对其他真菌毒素均无明显的交叉反应;对牛奶样品的检测结果与高效液相色谱-串联质谱法一致;且CLIA方法的试剂能在2~8℃稳定保存9个月以上。该研究建立的CLIA方法,具有灵敏、准确、快速和操作便捷等特点,检测时长仅30 min,尤其适用于大规模牛奶样品中OTA残留的快速筛查。
An automated chemiluminescent immunoassay(CLIA)based on acridinium ester-magnetic microspheres was established for the detection of ochratoxin A(OTA)in milk.Anti-mouse IgG antibodies were conjugated to magnetic microspheres and the OTA-BSA conjugates were labeled with acridinium ester.Different CLIA experimental conditions,including magnetic microsphere concentration,OTA antibody concentration,labeled OTA-BSA conjugate concentration,OTA antibody and labeled OTA-BSA dilution,reaction time,and sample pre-treatment conditions,were optimized.Under the established optimal conditions,the following results were obtained using the CLIA method:a limit of detection of 6.38 ng/kg,a half-maximal inhibitory concentration(IC50)of 31.40 ng/kg,a linear range of 11.23 to 99.20 ng/kg,and intra-and inter-assay coefficients of variation<8%.When determining OTA in milk samples,the limit of detection was 25.52 ng/kg,the IC50 was 125.60 ng/kg,the linear range was 44.92 to 396.80 ng/kg,and the addition recovery was 95.08%to 105.83%.The cross-reactivities with ochratoxin B and ochratoxin C were 6.52%and 24.57%,respectively,and there was no obvious cross-reaction with other mycotoxins.Milk sample detection values obtained using the CLIA method were consistent with those obtained using high-performance liquid chromatography-tandem mass spectrometry.Moreover,the reagents used for the CLIA method could be stored stably for more than 9 months at 2~8℃.In conclusion,the CLIA method established in this study is sensitive,accurate,rapid,and easy to operate.The detection time is only 30 min,and the method is particularly suitable for the rapid screening of OTA residues in large-scale milk samples.
作者
林健聪
梁科
柳彬
吴民富
罗林
雷红涛
沈玉栋
徐振林
LIN Jiancong;LIANG Ke;LIU Bin;WU Minfu;LUO Lin;LEI Hongtao;SHEN Yudong;XU Zhenlin(Guangdong Provincial Key Laboratory of Food Quality and Safety,College of Food Science,South China Agricultural University,Guangzhoug 510642,China;Dongshan Customs Comprehensive Technical Service Center of Fujian Province,Zhangzhou 363400,China;Department of Food Science,Foshan Polytechnic,Foshan 528137,China)
出处
《现代食品科技》
CAS
北大核心
2023年第3期313-322,共10页
Modern Food Science and Technology
基金
国家自然科学基金项目(32061160473)
广东省普通高校重点研究项目(2019KJDXM002)。
