黄曲霉毒素B_1的高灵敏时间分辨荧光免疫分析

Ultrasensitive time-resolved fluoroimmunoassay of aflatoxins B_1

采用时间分辨荧光技术建立了高灵敏的黄曲霉毒素B1(AFB1)间接竞争免疫分析法(AFB1-TRFIA)。以AFB1-BSA为免疫原免疫新西兰大白兔制备抗AFB1抗体;AFB1与辣根过氧化酶的联结物(AFB1-HRP)包被96孔板为固相抗原,与游离AFB1共同竞争有限的抗AFB1抗体;用稀土离子Eu3+标记的羊抗兔抗体进行示踪。该方法的灵敏度为0.01μg/L,测量范围为0.01—100μg/L,批内和批间变异分别为4.1%和6.8%,平均回收率为97.2%,与黄曲霉毒素B2的平均交叉反应为10%左右;黄曲霉毒素G2、赭曲霉毒素A、赭曲霉毒素B及HRP基本不干扰本方法对AFB1的检测。8条不同时间进行的AFB1-TRFIA的效应点值ED80、ED50、ED20分别为(0.07±0.01)μg/L、(0.63±0.02)μg/L和(12.5±0.47)μg/L。比较AFB1-TRFIA和AFB1-ELISA检测AFB1,两者的相关系数为0.917。研究表明,AFB1-TRFIA是目前报导的AFB1检测中最灵敏的方法,该分析方法稳定性好,可测范围宽,具有很好的应用前景。

The aim of this work is to provide a rapid, selective and very high sensitive method for the determination of aflatoxins B1（AFB1 using the indirect competitive time-resolved fluoroimmunoassay （TRFIA）. On the indirect competitive assay, aflatoxins B1-horseradish peroxidase （AFB1-HRP） is bound on the surface of a microtiter plate. AFB1 containing samples or standards and a rabbit anti- AFB1 antibody are given in the wells of the microtiter plate. Solid-phase bound and free AFB1 compete for the antibody binding sites, the secondary antibody, labelled with Eu^3＋ is used to enable detection. The luminescent enhancement solution contained mainly 2-naphthoyltrifluoroacetone. Results showed the AFB1 detection limit to be 0.01 μg/L for indirect competitive TRFIA formats. The 80%, 50%, 20% inhibition binding effect dose （ED80, ED50, ED20） of AFBj were （0.07±0.01） μg/L, （0.63±0.02）μg/L and （12.5±0.47） μg/L, respectively. The assay range was 0.01-100 μg/L. The cross reactivity with aflatoxins B2 was 10% and antibodies did not react with aflatoxins G2, ochratoxin A, ochratoxin B and HRP. The within-run and between-run CVs of the AFB1- TRFIA were 4.1% and 6.8%, respectively. The mean recoveries of samples were 97.2%. Both TRFIA and ELISA tests were applied to the quantitative measurement of AFB1 in the same samples, and the coefficient of correlation was 0.917. The present studies dearly show that the newly developed TRFIA could be applied to detect the AFB1 contamination in samples. The AFB1-TRFIA provides very high sensitivity and optimal range, and it will be useful to screen AFB1 contamination easily, simply and economically when the number of samples is large.