利用时间分辨荧光免疫层析法同时定量检测玉米中的伏马毒素B_(1)、B_(2)和B_(3)

Application of Time-resolved Fluorescence Immunoassay to Concurrently Quantify Fumonisin B_(1),B_(2)and B_(3)in Corn

建立了一种可同时定量检测玉米中伏马毒素B_(1)、B_(2)和B_(3)的时间分辨荧光免疫层析方法。利用活化酯法制备时间分辨荧光微球和伏马毒素抗体的偶联物,将伏马毒素抗原包被到硝酸纤维素膜上作为T线,通过优化不同T线包被条数,最终建立伏马毒素竞争性免疫层析方法。并对所建立方法的灵敏度、特异性、准确度、精密度和与国标方法分析结果的相关性进行评价。包被两条T线建立的检测方法检出限为107.68~168.28μg/kg,定量限为283.46~444.63μg/kg;与伏马毒素B_(1)、伏马毒素B_(2)和伏马毒素B_(3)的交叉反应率分别为100%、85.59%和72.72%,与其它5种常见的真菌毒素均无明显交叉;加标回收率范围88.37%~117.42%,变异系数小于10%;该方法与国标方法GB 5009.240-2016中规定的免疫亲和柱净化-高效液相色谱法(IAC-HPLC)检测结果的符合度在92.17%~107.21%之间。建立的时间分辨荧光免疫层析检测方法能满足对玉米中伏马毒素B_(1)、B_(2)和B_(3)的现场快速定量检测。

This study aimed to establish a time-resolved fluorescence immunoassay method for the simultaneous quantitative detection of fumonisin B_(1),B_(2)and B_(3)in corn.The EDC/NHS active ester method was used to prepare timeresolved fluorescent microspheres and fumonisin antibody conjugates.Fumonisin antigen was immobilized on a nitrocellulose membrane as the test line.By optimizing the number of test line,a competitive immunoassay method for detecting fumonisin was eventually established.Subsequently,the sensitivity,specificity,accuracy,precision,and correlation with the national standard method were evaluated for the established method.The detection limits of the established method by using two test line pattern were 107.68~168.28μg/kg,and the quantification limits were 283.46~444.63μg/kg.The cross-reactivity with fumonisin B_(1),fumonisin B_(2)and fumonisin B_(3)were 100%,85.59%and 72.72%,respectively,and no significant cross-reactivity was observed with five other common mycotoxins.The recovery rates ranged from 88.37%to 117.42%,and the coefficients of variation were lower than 10%.The conformity of the results obtained by the established method,as compared to the immunoaffinity column purification-high performance liquid chromatography(IAC-HPLC)method specified in the national standard GB 5009.240-2016,fell within the range of 92.17%to 107.21%.The established time-resolved fluorescence immunoassay method meets the requirements for rapid on-site quantitative detection of fumonisin B_(1),B_(2)and B_(3)in corn.