检测人血清中SARS冠状病毒IgG抗体的ELISA方法建立及其应用

为了建立方便、敏感和特异的SARS病毒血清学诊断方法,利用PQE30表达系统在大肠杆菌M15中分段高效表达了SARS病毒N蛋白.通过金属鏊合亲和层析纯化了目的蛋白N-1和N-2,Western blot结果显示,两个表达蛋白均具有较好的抗原性.然后将N-1和N-2蛋白共同包被,建立了检测人血清中SARS病毒IgG抗体的间接ELISA法.用此方法检测120例临床诊断为SARS的病人和244个不同年龄组正常人血清IgG抗体,结果120例SARS病人的第一份血清IgG抗体总阳性率为60.0%,发病第0～7、8～10、11～14、15～27和28天后的血清中,SARS病毒IgG抗体阳性率分别为0、11.1%、60.0%、60.5%和70.3%;而244份正常人血清检测结果均为阴性,包括100份14岁以下儿童血清也未发现假阳性.结果表明,利用大肠杆菌表达的N蛋白完全能够替代全病毒灭活抗原,所建立的间接ELISA方法简单,价格低廉,能保证生物安全,对SARS可疑病例的确诊和排除具有重要的实际应用价值,可用于SARS高危人群的血清流行病学监测,SARS疫情的控制和预防,以及SARS病毒蛋白功能的研究.

Establishment of an ELISA Assay to Detect the IgG Antibody of SARS Coronavirus and Its Application

Serological testing of SARS cases is very important for diagnosis and treatment of clinical patients,for control of SARS epidemics and the epidemiological study.A highly sensitive,specific and convenient indirect enzyme linked immunosorbent assay(ELISA)was established by using the N1 and N2 of nucleocapsid(N)protein of SARS coronavirus as the antigen.The N1 and N2 proteins were highly expressed independently using PQE30 expression system,and the target protein was purified by nickel chelating affinity chromatography.Western blot indicated that the N1 and N2 proteins had specific antigenicity with sera from confirmed SARS cases.Serum IgG antibody against N1/N2 protein of SARS coronavirus(SARS IgG)was tested in 120 clinically confirmed SARS cases and 244 normal subjects using this indirect ELISA.The first time collected serum samples from 120 SARS cases were drawn from different days of fever.The positive rates of SARS IgG of 120 sera were 0%,11 1%,60 0%,60 5%,70 3% at the 0-7,8-10,11-14,15-27 and more than 28 days after disease onset respectively,with the total positive rate of 60 0% SARS IgG was negative in the sera from 244 normal controls,including 100 children under 14 years old.In conclusion,we have established an indirect ELISA method using N1 and N2 of nucleocapsid protein of SARS coronavirus coated plate,which is highly specific and sensitive for the detection of SARS IgG in SARS cases.N1/N2 protein as antigen can replace inactivated whole SARS virus and can be used for clinical diagnosis,sero epidemiological investigation and protein functional study for SARS.