Development and Evaluation of ELISAs for Anti-hepatitis E Virus IgM and IgG Detection Based on Polymerized Recombinant Antigen
This paper focused on development and evaluation of anti-μ chain IgM ELISA and indirect ELISA for IgG antibodies against hepatitis E virus(HEV).Prokaryotic expressed polymerized recombinant HEV protein NE2 was used as antigen to develop two ELISA methods for anti-HEV IgM(E2-IgM)or IgG(E2-IgG)detection.Serial serum samples of twenty-nine HEV experimentally infected Rhesus monkeys were used to understand the dynamics of anti-HEV IgM and IgG detected by E2-IgM or E2-IgG as well as IgG detected by a generally used commercial kit(Genelabs, anti-HEV IgG,GL-IgG).Sera of 928 normal individuals and 510 clinical acute hepatitis patients were detected for anti-HEV IgM using E2-IgM,among them 200 patients were detected by E2-IgG at the same time.Sera of 5060 normal population were detected for anti-HEV IgG using E2-IgG.Sera of 119 non-A to C acute hepatitis cases were detected parallel for anti-HEV IgM using E2-IgM and a commercial IgM kit(GL-IgM).Two anti-HEV monoclonal antibodies which can capture HEV directly were used in blocking test for the evaluation of the specificity of E2-IgM and E2-IgG. The results showed the positive seroconversion rate of HEV by E2-IgM and E2-IgG methods in 29 HEV infected monkeys were both 100%,of which 75% were conversed within first 4 weeks post-infection,all earlier than the beginning time of ALT abnormality. Compared with 79.3% seroconversion rate by GL-IgG, 75% seroconversed within the first six weeks,much later than ALT abnormality. The seroconversion rate by E2-IgM lasted from two to fourteen weeks,with a mean of six weeks.Seroconversion by E2-IgG was a little later than E2-IgM, but none converted to negative at 70 weeks after infection. The average lasting time of GL-IgG was 18 weeks, only one monkey's IgG remained to be detectable by GL-IgG at 70weeks post infection. Among 928 normal people samples detected by E2-IgM,only 2 had OD value a little higher than 0.2.Two groups could be easily differentiated from 510 clinical acute hepatitis patients detected by E2-IgM ,one group was the same as detected in normal people with OD value lower than 0.2, the other group including 131 patients had OD value higher than 0.4,among them 109 samples were higher than 1.0. It suggested these two groups were corresponding to non-hepatitis E and hepatitis E patients.Patients with E2-IgM positive also had higher E2-IgG OD.Among 119 non-A to C acute hepatitis patients detected by E2-IgM and GL-IgM parallelly,57 were E2-IgM positive,including all the 29 patients who were positive by GL-IgM.Ten E2-IgM positive samples could be blocked by anti-HEV monoclonal antibodies(MAbs)from 66.4%～94.8%,with an average blocking rate of 86.6%.The OD values of 5060 normal population detected by E2-IgG formed a peak close to logarithm normal distribution with the mean of 0.022,and distributed evenly in samples with OD value higher than 0.4.Two groups also could be differentiated from 200 acute hepatitis patients,one with OD value lower than 0.2 as same as normal people,and the other with OD value higher than 1.0 formed another peak with peak OD value of 2.5,among the latter peak most were E2-IgM positive.Anti-HEV MAbs also could block E2-IgG effectively,and the blocking rate of Fab fragments of MAbs were the same as whole MAbs,which suggested that the blocking is epitope specific.The developed IgM and IgG ELISA methods have good specificity and sensitivity.IgM method is suitable for diagnosis of hepatitis E virus clinically infected,and the IgG method is suitable to diagnose hepatitis E virus infection in the past.