间接竞争酶联免疫分析法快速检测决明子中黄曲霉毒素B_(1)的研究

Rapid Detection of Aflatoxin B_(1)in Semen Cassiae by Indirect Competitive Enzyme-linked Immunosorbent Assay

目的:建立适用于中药决明子中黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))快速检测的间接竞争酶联免疫分析法(indirect competitive enzyme linked immunosorbent assay,ic-ELISA)。方法:首先采用棋盘滴定法优化抗原抗体最佳浓度,建立抑制曲线,然后以显色值和灵敏度为评价指标筛选样品的提取和稀释方法。对所建立的方法进行方法学考察,最后用于实际样品检测。结果:经过优化,确定样品的最佳提取溶剂为乙腈-甲醇溶液(90∶10,v/v),稀释溶液为甲醇-PBS溶液(10∶90,v/v),稀释倍数为20倍。方法的半数抑制浓度为0.078 ng/mL,线性范围为0.016~0.25 ng/mL,回收率为85.00%~109.2%,RSD为0.45%~9.08%。采用所建立的方法对25批决明子进行了检测,经液相色谱-串联质谱法确证,超过限量标准的样品的假阳性率小于5%。结论:本文所建立的ic-ELISA灵敏、简便,适用于决明子中AFB_(1)的快速筛查,但仍存在一定的假阳性率,后续还需要对产生干扰的基质因素进行研究并消除,进一步提高检测的准确度。

Objective:An indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)was established to determine aflatoxin B_(1)(AFB_(1))in Semen Cassiae.Methods:Firstly,the chessboard titration method was used to optimize the antigen and antibody concentration,and then the inhibition curve was drawn.Then,the sample extraction and dilution method for Semen Cassiaewas evaluated according to the matrix effect and sensitivity.The accuracy and precision of the established method were investigated.Finally,the proposed method was used to screen the real samples.Results:After optimization,it showed that the optimum extraction solvent for extraction is acetonitrile-methanol solution(90∶10,v/v).The diluted solution is the methanol-PBS solution(10∶90,v/v)with a dilution factor 20.The half-inhibitory concentration of the method was 0.078 ng/mL,and the linear range was 0.016~0.25 ng/mL.The recovery rates of the three spiked levels of 2.5,5.0,and 10.0μg/kg were 85.00%~109.2%with RSD of 0.45%~9.08%.A total of 25 batches of Semen Cassiae were detected using the established method.The liquid chromatography-tandem mass spectrometry method was employed for confirmation and it was found that the false positive rate of samples exceeding the limit standard was less than 5%.Conclusion:The ic-ELISA established in this study is sensitive and convenient,making it suitable for rapid screening of AFB_(1)in Semen Cassiae.However,there is still a certain false positive rate,and further research is needed to identify and eliminate interfering matrix factors to improve the accuracy of detection.