)j?m ELSEVIER Clinical and Diagnostic Virology 5 (1996) 167-179 Clinicaland Diagnostic Virology Artificial mosaic proteins as new immunodiagnostic reagents: the hepatitis E virus experience Howard A. Fields a'*, Yury E. Khudyakov ax, Michael O. Favorov a'c, Natalya S. Khudyakova a, Mian-er Cong a'd, Brian F. Holloway b, Stephen B. Lambert a, Danny L. Jue b aHepatitis Branch, Division of Viral and Rickettsial Diseases, National Center for InJeetious Diseases, Centers ./or Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, 1600 Clifton Rd., Atlanta, GA 30333, USA bBiotechnology Core l:aciliO, Branch, Scientific Resources Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA CThe D.I. lvanovsky Institute of Virology, Moscow 123098, Russia dlnstitute of Virology, Chinese Academy .[or Preventive Medicine, Beijing, China Received 10 October 1995; accepted 21 January 1996 Abstract Background: Naturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions. Objective: In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity. Study design: A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or fl-galactosidase. Results: Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific. A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera. A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA. Conclusion: An artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera. Keywords: Hepatitis E virus; Artificial mosaic protein; Oligonucleotides; Antigenic epitopes; Enzyme immunoassay * Corresponding author. Tel.: + 404 639 2335; fax: + 404 639 1563. Published by Elsevier Science B.V. PII S0928-0197(96)00218-8