伏马菌素B1免疫学检测方法的研究

Development of an immunologic method to detect the contamination with fumonisin B1 toxin in corn

目的制备抗伏马菌素B1(Fumonisin B1,FB1)单克隆抗体,在此基础上建立一种可用于定量检测玉米中FB1含量的间接竞争ELISA方法。方法人工合成免疫原BSA-FB1和包被抗原OVA-FB1,免疫BALB/c小鼠,采用杂交瘤细胞融合技术制备抗FB1的单抗,并对其特性进行鉴定。以OVA-FB1作为包被抗原,FB1为竞争抗原,两者与一定量的抗FB1单抗竞争反应,建立检测FB1的间接竞争ELISA方法,对玉米样品进行初步检测应用。结果获得了1株稳定分泌抗FB1单克隆抗体的杂交瘤细胞(命名为5H12-C6-C2)。对间接竞争ELISA方法进行反应条件优化后,建立了检测FB1的标准曲线,回归方程为y=1.113-0.313x,相关系数R2为-0.990,最适可测范围为10ng/ml^1000ng/ml,灵敏度为90.88ng/nl,最低检测限为7.83ng/ml,方法的批内和批间平均变异系数分别为3.24%和2.45%,样品添加平均回收率为94.32%。结论成功地制备了抗FB1特异性单抗,建立了FB1的间接竞争ELISA检测方法,为进一步研制FB1检测试剂盒奠定了基础。

To develop an indirect competitive ELISA method for the quantitative determination of fumonsin B1 toxin in corn,the artificially synthesized immunogen OVA-FB1 was used as the coating antigen and was immunized to BALB/c mice to prepare the specific monoclonal antibody against FB1 by hybridoma technique.One clone of the hybridoma cell stably secreting anti-FB1 antibody was obtained and named as 5H12-C6-C2.After optimization of the reactive condition,the standard curve for the indirect ELISA assay was established with the regression equation of y=⒈113-0.313 x（R^2=0.990）.The optimal detection range was 10 ng/ml to 1000 ng/ml,providing the sensitivity of 90.88 ng/ml,and the detection limit of 7.83 ng/ml.Meanwhile,the coefficients of variation of intra-and inter-assays were 3.24% and 2.45% respectively.The average recovery rate of FB1 in corn was found to be 94.32%.It is apparent this indirect competitive ELISA assay with specific monoclonal antibody can be used to detect the contamination of fumonisin B1 toxin in corn.