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  

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

作　　者：钟红[1] 马良[1] 张宇昊[1] 苏敏[1] 潘姝历 

机构地区：[1]西南大学食品科学学院,重庆400715

出　　处：《分析科学学报》2017年第2期195-200,共6页Journal of Analytical Science

基　　金：国家重点基础研究发展计划(973计划)(No.2013CB127803);国家自然科学基金(No.31301476)

摘　　要：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%。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（TAOBSA 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 1.48ng/mL.The regression equation was y=-21.65x＋53.68,R2=0.9941.The linear range was 0.06-35.95ng/mL.The sensitivity of the kit was 0.02ng/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.

关 键 词：细交链孢菌酮酸 人工抗原 多抗血清 酶联免疫法 

分 类 号：Q503[生物学—生物化学]