细交链格孢菌酮酸化学发光酶联免疫吸附分析方法

Indirect competitive chemiluminescence enzyme-linked immunoassay for tenuazonic acid

采用自制的抗细交链格孢菌酮酸(TA)多克隆抗体,建立了以辣根过氧化物酶催化鲁米诺-过氧化氢为发光体系的间接竞争化学发光酶联免疫分析方法定量检测谷物中TA毒素。通过研究选择最佳工作参数:反应缓冲液pH 6.4~7.4、包被浓度2.0μg/mL、封闭液1.0%OVA、抗血清稀释80000倍、竞争反应时间30 min、二抗稀释度为8000倍、发光底物125μL/孔。其线性回归曲线方程为y=49.82-20.14x,R^2=0.9966,半抑制浓度IC_(50)为0.973 ng/mL,最低检出限为0.010 ng/mL,线性范围0.032~30.244 ng/mL,在面粉和燕麦中的平均加标回收率为82.4%~96.1%和81.5%~93.4%,批内变异系数与批间变异系数均小于12%,与液质方法比较准确度无显著差异;与其他几种食品中的常见真菌毒素无交叉反应。可快速、准确、灵敏地测定谷物中TA检测。

An indirect competitive chemiluminescence enzyme linked immunosorbent assasy （ieCLEIA） based on HRP-Luminol-H202 system was developed for the detection of tenuazonic acid （TA） in grain samples. The CLEIA conditions were optimized as follows： the optimal buffer pH ranged from 6. 4 to 7.4, the concentration of coatigen was 2.0 μg/mL, the confining liquid was 1.0% OVA, the primary antibody dilution was 1：80000, the reaction time was 30 min, the IgG-HRP dilution was 1：8000 and the luminol substrate solution was 125 μL/well. Under optimized conditions, the regression equation was y = 49.82-20. 14x （ R^2 = 0. 9966 ） and the 50% inhibiting concentration （IC50） of the method was 0. 973 ng/mL. The linearity rang was 0. 032 - 30. 244 ng/mL. The limit of detection was 0. 010 ng/mL. The averaged recoveries of TA from wheat and oat were 82. 4% - 96. 1% and 81.5% -93.4%, respectively. The coefficients of variation of intra-assay and inter-assay were less than 12%. There was no significant difference of accuracy compare with UPLC-MS/MS, and no cross-reactivity with other mycotoxins. The method is rapid, accurate and sensitive for the determination of TA in cereals.