T-2毒素的高灵敏时间分辨荧光免疫分析

Ultrasensitive time-resolved fluoroimmunoassay of T-2 toxin

以时间分辨荧光免疫分析(TRFIA)方法建立快速灵敏的T-2毒素的全自动检测方法。采用T-2-BSA包被96孔板作为固相抗原,与游离的T-2竞争有限的抗T-2单克隆抗体,以Eu3+标记的羊抗鼠抗体示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立T-2-TRFIA。同时对这一方法的稳定性、灵敏度、回收率和特异性进行考核。此方法灵敏度为0.2μg/L,测量范围0.2～500μg/L,批内变异为7.5%,批间变异为12.7%,平均回收率为89.6%。与赭曲霉毒素A、黄曲霉毒素B1、牛血清白蛋白无交叉反应。10条不同时间进行的间接竞争T-2-TRFIA的效应点均值ED80、ED50和ED20分别为2.66,6.97,29.11μg/L。同时,用TRFIA和ELISA试剂盒同时检测T-2毒素,在ELISA和TRFIA产品的共同可测范围之内,两者的相关系数为0.926。

An indirect competitive time-resolved fluoroimmunoassay （TRFIA） was used to develop a rapid and high sensitivity method for the detection of T-2 toxin. In indirect TRFIA format, T 2-BSA was coated onto the microtitre plate and incubated with standard toxin and anti-T-2 antibody. A goal anti mice IgG： conjugated with Eu^3＋ was used to enable detection. The stability, sensitivity, mean recoveries, specificity of TRFIA method were tested. T-2 detection limit was 0. 2 μg/L for indirect competitive TRF1A formats. The assay range was 0.2-500μg/L. The within-run and between-run CVs of the T 2-TRFIA were 7.5A and 12.7% respectively. The mean recoveries were 89. 6%. The antibodies did not react with ochratoxin A, aflatoxin B1 and BSA. The 80%, 50% and 20% inhibition hinding effective dose（ED80, ED50, ED20） of T-2 were 2. 66, 6. 97, 29. 11 μg/L. Both T-2-TRFIA and T-2-ELISA test were applied for the quantitative measurement of T-2 in the same samples, and the coefficient of correlation was 0. 926. TRFIA method could be applied to detect the T-2 contamination. The T-2-TRFIA provides high sensitivity and optimal range, and it will be useful to detect T-2 toxin in food.