Establishment And Evaluation Of A Competitive Elisa For Detection Of Neutralizing Antibody Against Hepatitis E Virus

Hepatitis E virus (HEV) causes acute viral hepatitis worldwide; the highest incidence occurs in developing countries of Asia and Africa. The HEV is the causative agent of Hepatitis E (HE). HE is transmitted mainly by the fecal-oral route, usually through contaminated water, and occurs both in epidemic and in sporadic forms. The highest mortality occurs in infected pregnant women with case-fatality rates approaching 20% reported in many epidemics. HEV is a single-stranded positive-sense RNA virus overlapping open reading frames (ORFs). At present, HEV can be grouped into at least four major genotypes and several subtypes. This include genotypes I (several countries from Asia and Africa), II (Mexico, Nigeria), III (US, Argentina and Europe) and IV (China, Taiwan). Swine HEV in the US changed the epidemiology of HE. It raises the possibility of zoonotic spread of HEV. The study of distribution and transmission of HEV will be helpful to the control, diagnosis and treatment of HEV infection.Hepatitis E (HE) is endemic in China. However, an adequate level of information on the epidemiology of hepatitis E virus (HEV) has not been available so far because of the diagnostic methods. Now the diagnosis of HE mainly depends on immunodiagnostics. Problems remain both with the specificity of some assays, particularly when applied to seroepidemiological studies, and with the sensitivity of other assays for divergent strains of HEV and for the detection of past infection with HEV. The development of an enzyme immunoassay (EIA or ELISA) is now described using the ORF2 antigen expressed in E. coli, which provides a sensitive and specific assay for the detection and quantization of IgG anti-HEV in acute-and convalescent-phase sera. Fusion proteins of HEV ORF2 452~617aa (p166) of seven HEV strains clustered into different genotypes and subtypes were expressed in E. coli, and purified by the glutathione sepharose-4B affinity column, then identify the proteins by polyacrylamide gel electrophoresis (PAGE). At last we got seven pure recombinant proteins of the different genotypes and subtypes of HEV.Produced and purified monoclonal antibodies of 5G5,1G10,3G1.These three McAbs could react to all of the seven p166 recombinant proteins and they were all neutralizing antibody. Labeled antibody with HRP. Use the seven pure recombinant proteins and HRP 5G5 to establishment a competitive ELISA for detection of neutralizing antibody agaist HEV . Positive judging standard is> 50% of inhibition rate .To determine whether the ELISA could be used for detecting neutralizing antibodies,...