奶牛孕酮间接竞争ELISA检测方法的建立

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ELISA方法。结果]检测奶牛孕酮的间接竞争ELISA的最佳反应条件确定为:最佳包被浓度为100 ng/ml,碳酸盐缓冲液的包被效果较好,以4℃过夜+37℃2 h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=-1.844 4x-0.140 1(R2=0.981 7),检测范围为0~20 ng/ml,最低检测限为0.58 ng/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ELISA方法。

Establishment of Indirect Competitive ELISA for Detecting Progesterone in Dairy Cattle

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 optimum 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(R2=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.