Journal of Gastroenterology and Hepatology (2002) 17, S360–S364 Blackwell Science, LtdOxford, UK JGHJournal of Gastroenterology and Hepatology0815-93192002 Blackwell Science Asia Pty Ltd 17 17 Anti-HEV test A Obriadina et al. 10.1046/j.0815-9319.2002.00017.x Original ArticleS360S364BEES SGML Correspondence: Dr YE Khudyakov, Division of Viral Hepatitis, MSA33 National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Atlanta, GA 30333, USA. Email: yek0@cdc.gov CONFERENCE PROCEEDINGS A new enzyme immunoassay for the detection of antibody to hepatitis E virus A OBRIADINA,* † JH MENG,* ‡ T ULANOVA,* † K TRINTA,* § A BURKOV, † HA FIELDS* AND YE KHUDYAKOV* *Centers for Disease Control and Prevention, Atlanta, Georgia, USA, † NPO Diagnostic Systems, Nizhniy Novgorod, Russia, ‡ South-east University School of Medicine, Nanjing, China; and § Istituto Oswaldo Cruz, IOC—FIOCRUZ, Rio de Janeiro, Brazil Abstract Background and Aim: The purpose of the present study was to develop enzyme immunoassay (EIA) for the detection of IgG anti-hepatitis E virus (HEV) activity using two new recombinant proteins as antigenic targets, and to evaluate these EIA with the aid of statistical methods. Methods: Two proteins, a mosaic protein and pB166 containing region 452–617 aa of the ORF2 of the HEV Burma strain, were used to develop the new HEV EIA. This EIA was evaluated using several panels of serum specimens obtained from: (i) acutely HEV-infected patients; (ii) patients with non-A, non-C hepatitis; (iii) normal blood donors (NBD) from non-endemic countries; and (iv) experimentally infected chimpanzees. Results: A new HEV EIA was developed using two new recombinant proteins. This assay was able to detect anti-HEV activity in all specimens from acutely HEV-infected patients. When NBD were tested, more than 15% of specimens were found to be IgG anti-HEV positive. All NBD anti-HEV-positive spec- imens were tested with overlapping synthetic peptides spanning the entire HEV ORF2-encoded protein. More than 90% of the anti-HEV-positive NBD specimens immunoreacted with an average of 15 syn- thetic peptides derived from different regions of the HEV ORF2 protein. These data suggest that the HEV EIA is at least 90% specific in detecting remote HEV infections. Conclusion: The new HEV EIA developed in the present study is a highly specific diagnostic assay for the detection of anti-HEV activity in serum specimens obtained from different epidemiologic settings. © 2002 Blackwell Publishing Asia Pty Ltd Key words: enzyme immunoassay, hepatitis E virus, recombinant proteins, synthetic peptides. INTRODUCTION Hepatitis E virus (HEV) is a common etiologic agent of enterically transmitted non-A, non-B hepatitis. Out- breaks of HEV infection have occurred predominantly in developing countries of Asia and Africa, the central Asian republics of the former Soviet Union, and in the Middle East. 1–4 Hepatitis E virus is a currently unclassified non- enveloped virus 5 containing a single-stranded positive- sense RNA molecule of approximately 7.5 kb 6,7 Three open reading frames (ORF) were identified within the HEV genome: ORF1, which encodes for non-structural proteins; ORF2, which encodes for a putative structural protein(s); 7,8 and ORF3, which encodes for a small pro- tein with unknown function. The antigenic structure of this virus has been thoroughly studied. Several antigenic regions of diagnostic relevance were found within pro- teins encoded by ORF1, ORF2 and ORF3 by using overlapping synthetic peptides of different sizes 9–12 and recombinant proteins. 13–16 The cloning of the HEV genome and expression of recombinant proteins have allowed the development of enzyme immunoassays (EIA) for serological diagnosis of HEV. 13–15,17–22 The first experiments with recombi- nant proteins, however, demonstrated that not all HEV recombinant antigens are equally suitable for this pur- pose. It was found that synthetic peptides and some