同时检测玉米中黄曲霉毒素B_(1)和赭曲霉毒素A的时间分辨荧光免疫层析试纸条的研制

Development of a Time-resolved Fluorescence Immunochromatographic Strip for the Simultaneous Detection of Aflatoxin B_(1) and Ochratoxin A in Corn Samples

研究制备了一种可同时快速定量检测黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))和赭曲霉毒素A(ochratoxin A,OTA)的二联时间分辨荧光免疫层析试纸条。采用铕系时间分辨荧光微球分别标记AFB_(1)和OTA单克隆抗体,优化荧光微球活化pH值、标记的抗体浓度、荧光探针使用量、检测线包被原浓度、质控线羊抗鼠IgG浓度、样品前处理条件等因素。结果表明:检测AFB_(1)和OTA的IC_(50)分别为1.58、3.91μg/kg,线性范围分别为0.26~9.73、1.14~13.29μg/kg,肉眼检测限分别为3.70、5.55μg/kg;与黄曲霉毒素类似物交叉率为12%~62%,与赭曲霉毒素B交叉率为58%,与其他真菌毒素无明显交叉反应;选择玉米样品进行添加回收实验,2种毒素回收率在92%~103%之间,变异系数小于15%;对实际样品的检测与高效液相色谱法和市售胶体金试纸检测结果一致;且该试纸条可在4℃稳定保存5个月以上,稳定性较好。本研究所制备的时间分辨荧光免疫层析试纸条具有灵敏、准确、快速和简便等特点,非常适用于玉米等食品和农产品中AFB_(1)和OTA的现场快速筛查。

In this study,a time-resolved fluorescence immunochromatographic assay(TRFICA)for detecting aflatoxin B_(1)(AFB_(1))and ochratoxin A(OTA)simultaneously was developed.Europium-based time-resolved fluorescent microspheres were used to label anti-AFB_(1) monoclonal antibody and anti-OTA monoclonal antibody.Various factors such as pH for the activation of fluorescent microspheres,concentration of labeled antibodies,dosage of fluorescent probes,concentration of coating antigens for test line,concentration of goat anti-mouse IgG for quality control line,and sample pretreatment conditions were optimized.Under the optimal conditions,the assay showed half inhibition concentration(IC_(50))values of 1.58 and 3.91μg/kg,linear ranges of 0.26–9.73 and 1.14–13.29μg/kg,and visible limits of detection(vLOD)of 3.70 and 5.55μg/kg for AFB_(1) and OTA,respectively.The cross-reactivity rates of this method with aflatoxin analogues and ochratoxin B were 12%–62%and 58%,respectively,without cross-reactivity with other mycotoxins.The recoveries of the two toxins spiked in corn samples were 92%–103%,with coefficients of variation(CV)less than 15%.The results of this method for real samples coincided with those of high performance liquid chromatography(HPLC)and commercial colloidal gold strip.The TRFICA strip was stored at 4℃for more than 5 months with good stability.To sum up,this immunochromatographic strip is sensitive,accurate,rapid and easy to operate,and is very suitable for rapid screening of AFB_(1) and OTA in food and agricultural products.