ELISA方法检测牛奶中叶酸含量

摘要

Explored the establish of enzyme-linked immunosorbent assay (ELISA) method for milk folic acid detection and determine the initial content. Used a competitive enzyme immunoassay, checkerboard method to determine the optimal coating conditions, confinement, HRP labeled-antigen concentration etc., then evaluat-ed the performance and detect the folic acid content of 80 milk samples. The results showed: ①The optimal ELISA conditions:coated the folate antibody with the concentration of 2μg/mL, at the conditions of carbonate bufferand and 4℃, then blocked with 1%BSA, the working concentration of FA-HRP was 1μg/mL.②In this study, the detection limit was 2.22 ng/mL,the precision test CV<5%;the cross-reactivity with folic acid analogs was less than 0.1%;there was a significant correlation(r=0.960, P<0.05) with the control method in the val-ues of sample measurement;this method had a good recovery effect which was between 94.50%-108.00%;in this method, the sample matrix effects had no significant effect on the detection.③The concentration range of milk folic acid in the samples was12.55 ng/mL-68.72 ng/mL with an average of 37.93 ng/mL. This ELISA method could meet the testing of folic acid in milk.%探讨牛奶中叶酸检测的酶联免疫反应（Enzyme-Linked Immunosorbent Assay ， ELISA）方法建立及其叶酸本底含量测定。用竞争性酶联免疫，棋盘法确定最佳抗体包被条件、封闭条件、酶标抗原浓度等，并进行性能评价测定，检测80例牛奶样品中的叶酸本底含量。结果表明：①最佳ELISA条件为：叶酸抗体以2μg/mL浓度，碳酸盐缓冲液4℃过夜包被，用1%牛血清白蛋白（BSA）封闭，叶酸-辣根过氧化物酶标记物（叶酸-HRP）工作浓度1μg/mL。②该方法最低检测限2.22 ng/mL；精密度测试CV＜5%；特异性检测与叶酸类似物的交叉反应率均≤0.1%；本方法与对照方法具有良好的样本测值相关性（r=0.960，P＜0.05）；该方法加标回收率在94.50%~108.00%之间，回收效果良好；样品基质效应对该方法测值无显著影响。③牛奶样本叶酸本底含量范围为12.55 ng/mL~68.72 ng/mL，均值37.93 ng/mL。本研究建立的ELISA方法能够满足牛奶样品中叶酸含量的检测。