快速IgM抗体捕捉ELISA的建立及其在乙脑早期诊断中的初步应用

A Rapid lgM Antibody Capture ELISA and its Employ in the Early Diagnosis of Japanese Encephalitis

用辣根过氧化物酶标记的抗日本脑炎病毒(JEV)种特异性单克隆抗体为结合物,建立了一种快速IgM抗体捕捉酶联免疫测定法(RMAC-ELISA),并初步用于乙脑的快速诊断,取得了良好的效果。该法通过用鞣酸预处理微板,提高反应温度,在稀释液中加入PEG并提高NaCl浓度,用鸡蛋清代替牛血清白蛋白或鸡血清,用自来水代替PBS作为洗液,用快速漱洗3次代替3×5分钟洗涤,用TMBS代替OPD底物,用酶标单克隆抗体代替多克隆结合物等措施,加快了反应速度,缩短了反应时间,简化了操作步骤,提高了敏感性,保证了特异性。从微板预处理至出结果,仅需1小时。由于该法具有简便、快速、特异、敏感等特性,不仅可以用于乙脑的早期诊断及实验室研究,还有可能推广到其它的免疫学检测方法中去。

A rapid IgM antibody capture (RMAC) ELISA using enzyme labeled one ofthe species specific McAb 2H_4 as tracer was studied for the early diagnosis.The antibody thus obtained was confirmed specific, because it was destructableby 2-ME, could be blocked by JEV antigen, and had no cross reactivity with HFRS virus or adenovirus IgM antibody positive sera, as well as was notinterfered by RF. 10 serum specimens were tested for JEV-IgM by conventionalMAC-ELISA and RMAC-ELISA simultaneously, and the Iatter one was moresensitive. By pretreating the microplate with tannic acid, rapid washing withtap water instead of three times of 5 minutes with PBS after each incubations,reacting 10 minutes at 40℃ with PEG, egg white and higher concentration ofNaCl in PBS instead of 1 hour at 37℃ with only PBS for each of the incub-ations, the RMAC-ELISA not only had higher sensitivity, but also could befinished in less than 1 hour. Since the RMAC-ELISA was a simple, conveni-ent, rapid and sensitive method, it could be employed in ordinary virology labfor both basic reasearoh and early diagnosis of JEV infection, and it could beextended to other immunoassays either.