检测大肠杆菌菌体蛋白的夹心ELISA试剂盒的建立及应用研究  

Establishment and application of sandwich ELISA kit for detection of E. coli cell proteins

作　　者：李琦[1] 张葵[1] 庄然[1] 张赟[1] 宋朝君[1] 徐竹蔚[1] 陈丽华[1] 杨琨[1] 金伯泉[1] 

机构地区：[1]第四军医大学免疫学教研室,西安710032

出　　处：《国际生物制品学杂志》2010年第6期281-284,共4页International Journal of Biologicals

基　　金：国家科技支撑计划（2006BAF07801）

摘　　要：目的建立特异、敏感的检测大肠杆菌菌体蛋白的夹心ELISA试剂盒。方法采用大肠杆菌菌体蛋白免疫家兔，制备得到高效价抗菌体蛋白抗血清。将经饱和硫酸铵盐析、阴离子交换层析和亲和层析纯化的兔抗大肠杆菌菌体蛋白特异性多克隆抗体作为包被抗体和检测抗体，用生物素标记检测抗体，并加入辣根过氧化物酶标记的亲和素。结果建立的大肠杆菌菌体蛋白夹心ELISA检测方法的敏感性为0．32μg／L，检测范围为1～100μg／L，与国际同类商品化试剂盒相当。此法具有良好的稳定性，其批内变异系数小于7．7％，批间变异系数小于6．2％。结论建立了特异、敏感、稳定的检测大肠杆菌菌体蛋白的夹心ELISA试剂盒，为生物制品中残留大肠杆菌菌体蛋白的质量控制提供了一种重要的检测方法。Objective To establish a highly specific and sensitive sandwich ELISA kit for detection of E. coli cell proteins. Methods E. coli cell proteins were used for immunization of rabbits and high titer antisera were obtained. The polyclonal antibodies （PAbs） against E. coli cell proteins were purified by saturated ammonium sulfate salting-out, anion exchange chromatography and affinity chromatography. The purified PAbs were used as coating antibodies, and biotin-labeled PAbs plus horseradish peroxidase-conjugated avidin were used as detecting antibodies. Results The sensitivity and detection range of the sandwich ELISA system for detection of E. coli cell proteins were 0.32 i.~g/L and 1-100 txg/L, respectively, and equal to the similar commercial kits in the world. This ELISA kit had good stability, and its intra- and inter-assay variation coefficients were less than 7.7% and less than 6.2%, respectively. Conclusions The sandwich ELISA kit with high specificity, sensitivity and stability is established successfully and provides an important method for quality control of residual E. coli cell proteins in biological products.

关 键 词：大肠杆菌 酶联免疫吸附测定 蛋白质 多克隆抗体 

分 类 号：R[医药卫生]