牛奶中孕酮直接竞争ELISA检测方法的建立

Establishment of direct competitive ELISA method for the detection of progesterone in milk

采用直接竞争ELISA方法检测牛奶中孕酮含量。采用碳化二亚胺法，应用辣根过氧化物酶（HRP）标记11-α羟基孕酮琥珀酸酯（11α—OH—P4-HS），制备孕酮酶标抗原；酶标抗原与牛奶样品中的孕酮共同竞争结合固相包被的孕酮单克隆抗体，建立直接竞争ELISA反应体系。试验结果表明，最佳抗体包被浓度为1：2000，最佳酶标抗原工作浓度为1：16000，建立的标准曲线为y=-2．4598x＋0．999（R^2=0．996），牛奶中孕酮检测范围0．12-40ng／mL，最低检测量为0．12ng／mL，批内和批间变异系数分别为1．63％和2．49％。成功建立了可用于牛乳中孕酮快速检测的直接竞争ELISA方法。

The objective of this study was to detect the content of milk progesterone using direct competitive ELISA method. Enzyme-Labelled progesterone Antigen was prepared according to carbodiimide method and 11-α-OH demi-succinate（11α-OH-P4-HS） was labeled by HRP. Both enzyme-labelled antigen and milk sample with progesterone as competitor were incubated with solid-phase coated with anti-progesterone monoclonal antibody. The results showed that optimum coating antibody content was 1 ： 2 000, optimum enzyme-labelled antigen was 1 ： 16 000, institutional standard curve was y=-2. 4598xq-0. 999（R^2 =0. 996）, detection range was from 0.12 to 40 The inter-assay and intra-assay coefficient variation ng/mL and the lowest detectable value was 0.12 ng/mL. was 1.63% and 2.49% respectively. So, a fast quantitative detection competitive ELISA method for milk progesterone was established.