细交链格孢菌酮酸间接竞争酶联免疫检测方法及配套试剂盒的研制

建立了间接竞争酶联免疫吸附法检测食品中细交链格孢酮酸(Tenuazonic Acid,TA)残留,并研制快速检测试剂盒。采用肟化法对TA进行衍生化,活泼酯法偶联半抗原和载体蛋白得到人工抗原TAO-BSA和TAO-KLH,以TAO-KLH作为免疫原免疫雌性Balb/c小鼠制备特异性抗体。对包被浓度、包被时间、封闭液类型、抗体工作浓度、二抗稀释度、底物显色时间等参数进行研究,建立了TA残留间接竞争酶联免疫检测方法并进行了配套试剂盒的研制。该试剂盒半抑制浓度Ic_(50)为1.48ng/mL,检测线性范围为0.06~35.95ng/mL(R~2=0.9941);检测限为0.02ng/mL;加标样品平均回收率大于88.50%;试剂盒的批间和批内平均变异系数分别为2.84%和9.49%,与食品中常见真菌毒素交叉反应率均小于1%。

Study on Indirect Competitive Enzyme Immunoassay and Rapid Detection Kit for Tenuazonic Acid

An indirect competitive enzyme immunoassay and rapid detection kit with polyclonal antiserum was established for rapid screening of tenuazonic acid(TA) in food.Two TA-amine conjugates(TAO-BSA and TAO-KLH) were synthesized by using carboxymethoxylaminc hemihydrochloride to derivatize TA and coupling the products with carrier protein through the active ester method.Female Balb/c mouse were subcutaneously immunized with the TAO-KLH for obtaining mouse polyclonal antiserum.The effecting factors of icELISA were optimized.Under optimized conditions,the 50％ inhibiting concentration was 1.48 ng/mL.The regression equation was y =-21.65xc+ 53.68,R2 =0.9941.The linear range was 0.06-35.95 ng/mL.The sensitivity of the kit was 0.02 ng/mL,and the recovery was higher than 88.50％.The coefficient of variation of intra-assay and inter-assay was 2.48％ and 9.49％,respectively.And no cross-reactivity with other mycotoxins was observed.