间接竞争ELISA方法测定水中2,4-D的研究

Development of Indirect ELISA for the Determination of 2,4-D in Water

以2,4-D-BSA为包被抗原,采用自行制备的2,4-D单克隆特异性抗体6D11建立了水中2,4-D的间接竞争 ELISA检测方法．本研究比较了包被抗原2,4-D-BSA浓度分别为240ng·mL-1、120ng·mL-1和60ng·mL-1的反应体系和竞争反应时间为60min和15min的间接竞争ELISA剂量-反应曲线,确定了当包被抗原浓度为60 ng·mL-1、竞争反应时间为15min时,剂量-反应曲线的IC50值较低．采用上述实验条件分别测定了由PBS缓冲溶液、饮用水、清华大学地下水和圆明园福海地表水配制的2,4-D标准溶液的剂量．反应曲线,发现实际水样的基质效应对检测结果的影响较大;采用实际水样和PBS缓冲溶液配水在含有5％乙醇的PBS缓冲体系中反应的方法,基本上消除了基质效应对检测结果的干扰．采用上述优化试验条件,测定2,4-D浓度分别为0．5mg·L-1、0．125mg·L-1和0．03mg·L-1的加标样品,测定数据的准确度符合痕量有机污染物定量检测对准确度的要求,但是平行样品测定数据之间的变异系数较大,需要进一步改进检测方法,用于实际水样的检测．

The indirect competitive ELISA was developed for the determination of herbicide 2,4-D in water sample with selfmade specific monoclonal antibody 6D11 against 2,4-D. The dose-response curves were plotted when coating antigen 2,4-D- BSA concentration respectively were 240ng·mL^-1, 120ng·mL^-1 and 60ng·mL^-1. Lower ICso value was taken when coating antigen concentration was 60ng·mL^-1. The dose-response curves were also plotted when competitive step respectively was within 60rain and 15min. Lower ICso value was taken when the competitive step was withinl5min. 2,4-D concentration in PBS, drinking water, ground water of Tsinghua University and surface water of Fuhai Lake in Yuanmingyuan Park were detected using above optimum reaction condition. It was found that the matrix effects interfered in detection precision of water samples. The matrix effects were basically eliminated using PBS buffer of 5% ethanol. 2,4-D concentration of 0.5mg·L^-1, 0.125mg·L^-1 and 0.03mg·L^-1 in water samples were determined at above optimal reaction conditions. The recoveries of 2,4-D varying from 70% to 130%, which is approximately according to the precision standard of trace organic pollutants. But the coefficients of variation （CV） were relatively larger. The established immunoassay should be improved in order to practical determination of 2,4-D in water samples.