酶联免疫吸附法测定麻疯树核糖体失活蛋白

An Enzyme-linked Immunosorbent Assay for Determination of Ribosome-inactivating Protein from Jatropha curcas L.

建立了一种检测麻疯树核糖体失活蛋白(Curcin)的简便、快速的酶联免疫吸附测定法(ELISA).ELISA条件优化试验结果显示:最佳条件为costar酶标板在紫外线下照射45 min后进行包被;抗原最适稀释度为1:640倍(2.5μg/mL);curcin抗血清最适稀释度为1:12 800倍;2%封闭用山羊血清封闭120 min;二抗最适稀释度为1:5 000倍.在此试验条件下检测curcin含量,得到回归方程y=31.722x+106.88,相关系数R2=0.993 3.Curcin浓度在0.08～11μg/mL之间,曲线呈良好的线性关系.本方法的最低检测限为0.08μg/mL.高、中、低3个浓度的添加回收率分别为95.56%、102.27%、98.40%,平均回收率为98.74%.板内和板间的平均变异系数分别为4.78%和6.71%.Curcin抗血清与2种大戟科同类蛋白均无交叉反应.利用本方法成功检测了麻疯树种仁及脱毒饼粕中的curcin含量.

A indirect ELISA method was established to detect curcin from Jatropha curcas L.Through optimizing the conditions of ELISA,the best results from this study indicated that the costar ELISA plates were coated after irradiating ultraviolet for 45 min,the optimum dilution of antigen was 1/640（2.5 μg/mL）,the optimum dilution of antiserum of curcin was 1/12 800,the coster ELISA plates was closed 120 min by 2% goat serum,and the optimum dilution of the second antibody was 1/5 000.In these experimental conditions,curcin was detected and the regression equation y=31.722 x ＋ 106.88 was obtained,with the correlation coefficient being R2=0.993 3.The standard curve of indirect ELISA of curcin showed good linear relationship between the concentrations of 0.08～11 μg/mL.The lowest concentration for detecting curcin was 0.08 μg/mL.The recovery rates of curcin of high,medium and low concentrations were 95.56%,102.27%,98.40%,with the average rate being 98.74%.The average variation coefficients of intra-assay and inter-assay were 4.78%,6.71%,respectively.The specificity tests were done with two kinds of similar proteins.The antiserum of curcin had no cross-reaction.The contents of curcin in kernel and dreg of J.curcas were detected successfully.