酶联免疫吸附分析方法(ELISA)检测血浆中糖基化终极产物

目的建立竞争性ELISA方法，测定血液中的高级糖化终产物的含量。方法对兔及牛血浆白蛋白进行体外葡萄糖修饰，得到糖基化终极产物抗原；用兔此抗原免疫新西兰大白兔得到与其特异结合的抗体。结果1．经过TNBS方法及聚丙稀凝胶电泳鉴定修饰抗原，发现修饰前后蛋白携带自由氨基数至少35％已被葡萄糖修饰且分子量也发生变化。2．免疫获得抗糖基化终极产物的多克隆抗体，并鉴定了此多抗只抗葡萄糖修饰的蛋白而不抗载体蛋白。3．用葡萄糖修饰的蛋白及其多克隆抗体制成ELISA试剂盒，批间、批内变异系数分别为5．8％和9．8％，灵敏度为0．5u／ml。结论得到检测血浆糖基化终极产物的ELISA试剂盒。

ELISA detects the plasma advanced glycation end products

Objectives To establish the ELISA method to detect the plasma advanced glycation end products.Methods 1. AGEs were prepared in vitro by incubating rabbit(AGEs-RSA)and bovine albumin(AGEs-BSA)with glucose;Polyclonal Anti-AGEs antibodies were generated by injecting subcutaneously AGEs-RSA into New Zealand white rabbit.Results 1. At least 35% of free amino groups of albumin was modified by glucose by using TNBS and Molecular weight before and after modifying have changed by using PAGE methods.2. The polyclonal Anti-AGEs antibodies were specifically against AGEs-proteins not the carrier proteins.3. The plasma AGEs levels was measured with ELISA by using the generated antibodies and modified albumin as standard and the inter-assay and intra-assay were 5.8% and 9.8%,respectively.Conclusion A stable ELISA method for detecting the plasma AGEs has been established.