奶牛孕酮间接竞争ELISA检测方法的建立

Establishment of Indirect Competitive ELISA for Detecting Progesterone in Dairy Cattle

[目的]为孕酮检测试剂盒的研制奠定基础。[方法]采用二环己基碳化二亚胺法制备检测抗原，建立了检测奶牛孕酮含量的间接竞争EHSA方法。[结果]检测奶牛孕酮的间接竞争EHSA的最佳反应条件确定为：最佳包被浓度为100ng／ml，碳酸盐缓冲液的包被效果较好，以4℃过夜＋37℃2h佳，最佳封闭液为2％山羊血清，酶标抗体的工作浓度为0．13μg/ml。建立的标准曲线方程为y=-1．8444x-0．1401（R^2=0．9817），检测范围为0～20ng／ml，最低检测限为0．58ng／ml。该标准曲线的批内变异系数和批间变异系数分别为2．22％和2．37％。[结论]该研究建立了定量检测奶牛孕酮的间接竞争EHSA方法。

[ Objective] The aim of the research was to lay the foundation for developing the reagent kit of detecting progesterone. [ Method] The detection antigen was prepared by using dicyclohexyl carbodiimide method. And an indirect competitive ELISA for detecting progesterone content in diary catle was set up. [ Result ] The optimum reaction conditions of indirect competitive ELISA for detecting progesterone in dairy cattle were as follows： the optimmn coating concn, was 100 ng/ml, the coating effect of carbonate buffer was better with that of staying over a night at 4 ℃ and coating for 2 h at 37 ℃ being best, the optimum sealing liquid was 2% goat serum and the working concn, of enzyme-labelled antibody was 0.13 μg/ml. The established standard curve equation was y = - 1. 844 4x - 0. 140 1 （ R^2 = 0.981 7）, with the detection range and the lowest detection limit of 0 - 20 ng/ml and 0.58 ng/ml resp. The intra-assay and inter-assay coefficient variations of this standard curve were 2.22% and 2.37 % resp. [ Conclusion ] In this research, the indirect competitive ELISA for detecting progesterone in dairy cattle quantitatively was established.