Establishment And Preliminary Application Of ELISA Detection Method For Hepatitis E Virus IgG,IgM And IgA Antibodies

PurposeTo establish and evaluate an indirect method to test Hepatitis E Virus(HEV)IgG、IgM、IgA antibodies,evaluate the established indirect ELISA methods for anti-HEV IgG,IgM and IgA detection,detect the infection of Hepatitis E virus among Blood donors in Xishuangbanna Autonomous Prefecture,and preliminarily evaluate the anti-HEV IgA test the role of HEV screening.MethodsCarrying out Tn-5 cell culture and serum domestication,infect recombinant HEV baculovirus,extract and purify HEV VLP(virus-like particles),and verify with SDS-PAGE and Western-blot.Collecting 2364 whole blood samples from Yunnan province and store under 80℃ after centrifuging.A total of 1864 blood donor samples were detected by the method described in research and Wantai HEV antibody kit,and anti-HEV IgG,IgM,IgA negative samples and positive samples were screened for subsequent method establishment.To optimize the reaction conditions of the indirect ELISA method,establish the indirect ELISA method for detection of hepatitis E virus IgG,IgM and IgA antibodies and conduct performance evaluation.Using the method established in this article to detect 500 blood donor plasma samples from Xishuangbanna Autonomous Prefecture,Yunnan,and count the positive rates of anti-HEV IgG,anti-HEV IgM as well as anti-HEV IgA,calculate the recent HEV infection rate(positive for any anti-HEV IgM/IgA)and HEV seroprevalence(anti-HEV IgG/IgM/IgA any positive),analyze the risk factors related to HEV infection,and evaluate the role of anti-HEV IgA testing in HEV screening.ResultsTn-5 cells can multiply well in serum-free medium,and there is no contaminant protein after purification,and the concentration of HEV VLP which were extracted and purified from the culture medium is 3.95mg/mL.Both of the specificity and sensitivity of the established indirect ELISA methods for hepatitis E virus IgG and IgM antibodies were 100%.The specificity and sensitivity of the established indirect ELISA methods for hepatitis E virus IgA antibody was 95%and 100%.When anti-HEV IgG,IgM and IgA were detected,there was no statistical difference in 0D value after anti-HCV,anti-HIV,anti-TP,anti-HSV-2,HBsAb,HBeAb,HBcAb positive samples;lipemia,hemolysis and ALT were added(P>0.05).The coefficients of variation of intra-assay and inter-assay repetition are less than 10%.The positive rates of anti-HEV IgG,IgM and IgA in blood donors in Xishuangbanna Prefecture were 18%(90/500)、5.6%(28/500)and 2.6%(13/500),and the recent HEV infection rate was 1.8%(9/500),and the seroprevalence of hepatitis E is 19.8%(99/500).If only anti-HEV IgM single positive was used as the criterion of HEV recent infection,7 cases of acute infection were found in 500 blood donors,but if combined anti-HEV IgA(IgG negative and IgM/IgA positive)were used as the criterion of HEV recent infection,9 cases of acute infection were found in 500 blood donors.Of the 500 blood donors,13 were anti HEV IgA positive,2 were anti HEV IgG negative,10 were anti HEV IgM negative,and 2 were both anti HEV IgG and IgM negative.The coincidence rate of the method in this article and the Wantai kit for the anti-HEV IgG test results of blood donors is 84.57%;the coincidence rate of the anti-HEV IgM test results is 96.28%,and the consistency is good。The methods in this article and the literature methods are effective for blood donors and the coincidence rate of anti-HEV IgA test results was 95.21%.ConclusionIn this study,three indirect ELISA methods for detecting hepatitis E virus IgG,IgM and IgA antibodies were established by using the recombinant protein of HEV ORF2(Open Reading Frame 2)region(aa122-660)expressed by Insect Baculovirus Expression Vector System as the coating antigen.They can be used to detect human HEV IgG antibody,human HEV IgM antibody and human HEV IgA antibody,respectively.The three established indirect ELISA methods have high specificity and sensitivity,showed a strong ability to against interference like lipemia,hemolysis,ALT and other pathogens.HEV antibody positive is common among blood donors in Xishuangbanna Autonomous Prefecture of Yunnan Province,some of which are recent infections,posing a threat to the safety of blood transfusion.Human HEV IgA antibody indirect ELISA method combined with human HEV IgM antibody detection can increase the detection rate of recent HEV infections.It is very important for the clinical diagnosis and epidemiological screening and prevention of HEV infection,but HEV still needs to be collected in the follow-up Samples from patients diagnosed with acute infections for clinical verification.