人红细胞胰岛素受体自身磷酸化的酶联免疫吸附测定

ELISA method for autophosphorylation of insulin receptor from human erythrocytes

目的建立人红细胞胰岛素受体自身磷酸化测定法。方法应用合成的磷酸酪氨酸钥孔帽贝血蓝蛋白（ＰＹＫＬＨ）免疫家兔得到抗血清，将纯化的抗磷酸酪氨酸抗体作为第一抗体，羊抗兔Ｉｇ酶结合物（辣根过氧化物酶标记）作为第二抗体，建立双抗体夹心酶联免疫吸附测定法。结果批内变异系数为８．４％，批间变异系数为９．０％。２０例健康人的红细胞ＩＲ的自身磷酸化活性，测定值为０．１５０±０．０２２ｎｍｏｌＰＹ／ｍｇ受体蛋白。在体外，胰岛素在１０－６ｍｏｌ／Ｌ时对自身磷酸化达到最大激活。结论用酶免法测定人红细胞胰岛素受体灵敏、特异、简便，为胰岛素作用机制的理论研究、胰岛素受体基因突变的检测和糖尿病及其它胰岛素抵抗综合症的病因及治疗的探讨提供了有效的方法。

Objective To establish a method of detecting autophosphorylated insulin receptor from erythrocytes. Methods Phosphotyrosine was first combined with hemocyanin from keyhole limpets (KLH). Then, rabbits were immuned with the complete antigen and antisera were gained. The ELISA system consisted of purified antiphosphotyrosine antibody as first antibody and sheep antirabbit lgG HRP as second antibody. Results CV within the batch was 8.4%. CV between the batches was 9.0%. The value for autophosphorylation of insulin receptor from erythrocytes in 20 normal individuals was 0.150±0.022nmoL PY/mg receptor protein. In vitro, autophosphorylation of insulin reached maximum activation at 10 -6 mol/L insulin. Conclusion The ELISA method is sensitive, specific and simple for autophosphorylation of insulin receptor. It provides a useful tool for theoretical research of mechanism of insulin action, detection of gene mutation of insulin receptor and exploration of cause and care for insulin resistance.