DEVELOPMENT OF AN in-house ELISA FOR THE SEROLOGICAL DIAGNOSIS OF HEPATITIS E VIRUS INFECTION

ARCE, LORENA ; VIZOSO PINTO, M. GUADALUPE

Buenos Aires

Congreso; IV International Clinical Virology, Symposium and Advances in Vaccines; 2016

CEMIC

BACKGROUND: Sporadic hepatitis E virus (HEV) infection associated with livestock in industrialized countries is growing, chronic HEV infection has been detected in immunosuppressed individuals (patients with AIDS or haematological malignancies, and transplant recipients), HEV transmission through blood transfusion has been reported, and HEV is the major cause of acute hepatitis in developing countries of Africa, Asia and the Middle East: therefore, the National Institutes of Health has classified Hepatitis E as an emerging disease. In Argentina, epidemiological data on HEV infection occurrence and prevalence are scarce, the disease is believed to be rare, specific testing is not usually offered, and serological diagnostic tests are not produced. Wassaf et al. detected HEV in patients and wastewater from Córdoba, a midland province in Argentina, underscoring the need to have such tests locally and readily available. Objective: to develop an in-house ELISA using a recombinant ORF2 of HEV genotype 3 protein produced in E. coli. Material and methods:_the capsid protein ORF2 encoded in pETG-N-His was expressed in E. coli Rosetta. The His-tagged protein was purified using NiNTA under native and denaturing conditions. Purity was checked using SDS-PAGE and Western_blotting. Different recombinant protein concentrations were tested as capture antigen for coating 96-well ELISA plates. Gelatine, bovine serum albumin (BSA), and milk were assayed as blocking agents. Several dilutions of positive and negative sera and secondary HRP-antibody were tested for optimization. Results: ORF2 of GT3 HEV was primarily expressed in the cytoplasm, and to a lesser extent in inclusion bodies. The recombinant protein was successfully purified on NiNTA as analyzed by SDS-PAGE and Western_blotting. Blocking with gelatine or BSA yielded similar and optimal results, whereas milk yielded a higher background in negative samples probed with a negative serum with less than 0.2 absorbance units at A450 nm. The optimal dilutions were: 1. Serum, 1:80; 2. Secondary antibody (anti-IgG-HRP), 1:2000. Conclusions: We provide a prototype ELISA for detecting specific anti-HEV antibodies, and plan to evaluate a larger panel of characterized sera to determine its sensitivity and specificity.