一种新型玉米赤霉烯酮的定量检测方法

A New Method for the Zearalenone Quantitative Detection

采用间接竞争法原理和液相芯片技术平台,选用玉米赤霉烯酮单克隆抗体(Monoclonal Anti-ZEN)对玉米赤霉烯酮(ZEN)定量检测方法进行探索。将ZEN-BSA与36﹟微球偶联,同时加入待检的游离的ZEN小分子和其标记有生物素的ZEN单克隆抗体,偶联抗原和目标抗原竞争结合生物素标记抗体,通过报告分子SA-PE在532 nm波长的激光下,即可检测出报告分子发出的荧光强度,荧光强度与待检ZEN的量成反比。该研究优化了液相反应体系中ZEN单克隆抗浓度、确定了ZEN单抗临界饱和浓度以及抗原抗体最佳孵育时间,其中ZEN单抗临界饱和浓度为2.0μg/mL,偶联抗原和抗体最佳孵育时间为3h。研究所建立的ZEN液相芯片定量检测方法,以国际通用的IC50作为衡量标准,其检测灵敏度为0.005 4μg/mL,线性范围为0.000 8～0.04μg/mL。应用该方法对脱脂牛奶和全脂牛奶进行加标回收检测,平均回收率均大于75%。该方法简化了样品的纯化和分离步骤,经过实际样品的检测,证明方法灵敏、稳定、快速、简便,适用于大量样本的检测。

The quantitative detection of Zearalenone（ZEN） was investigated on the microsphere array technology platform and indirect competition theory. In this research, Monoclonal Anti-ZEN was chosen and labeled by biotin. ZEN-BSA was coupled with 36# microsphere, then it was added in the 1.5mL centrifuge tube with the free target antigen and the biotin labeled Monoclonal Anti-ZEN. Botin labeled Monoclonal Anti-ZEN was competitive bound by the coupling antigen and the free target antigen, and the target antigen was detected by the report molecule SA-PE which was stimulated fluorescence at 532nm laser light. The fluorescence intensity was inversely proportional to the concen- tration of the free target antigen. Through optimizing the concentration of the critical saturated Monoclonal Anti-ZEN and determining the incubation time of antigen and antibody, the quantitative detection of ZEN was established eventually. In this research, the critical saturated concentration of Monoclonal Anti-ZEN was 2.0 μg/mL, the best incubation time of antigen and antibody was 3h. As referring of the international current standard of IC50, the results showed that the sensitivity was 0. 0054 μg/mL, the detection range of the standard curve was 0. 0008 - 0.04 μg/mL. By applying the research method in skim milk and full milk contamination testing, the recovery rate of the two kinds of milk was both greater than 75%. The procedure of purification and separation of the sample were simplified by using this method. Overall, the quantitative analysis of ZEN using the microsphere array technology is sensitive, stable, rapid, and simple, and it is applicable for mass sample testing.