Artificial mosaic proteins as new immunodiagnostic reagents: the hepatitis E virus experience.
ABSTRACT 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.
In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity.
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 beta-galactosidase.
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.
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.
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ABSTRACT: Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive for detecting anti-HEV activity in the sera of patients with acute hepatitis E, convalescents, and among healthy individuals.Journal of Virological Methods 02/1994; 46(2):237-50. · 1.90 Impact Factor
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ABSTRACT: Hepatitis E virus (HEV) is thought to be a cause of enterically transmitted non-A, non-B (ET-NANB) hepatitis. Waterborne epidemics have been recorded in many developing countries, mainly affecting young-to-middle-aged adults; sporadic infection and overt illness in children are rare. However, a convenient and sensitive diagnostic test for HEV infection is not yet available. We now report the use of a solid-phase enzyme-linked immunoassay (ELISA) that detects IgM and IgG antibody to HEV. In a prospective study of endemic acute hepatitis during 1986 in rural Benha, Egypt, 15 (42%) of 36 children with NANB hepatitis (from whom convalescent-phase sera were available every 3 months to 9 or 12 months) were positive for anti-HEV-IgG by ELISA. Of 20 sera from healthy Benha children (controls), 5 (25%) were also positive for anti-HEV-IgG. When evaluated for anti-HEV-IgM, 6 of the 15 IgG-positive children, but none of the controls, were IgM positive and were thus regarded as having confirmed acute HEV infections. These 6 cases together with 2 presumptive cases (IgM negative, IgG seroconversion from positive to negative) presented sporadically over 9 months. This ELISA is a convenient method for the diagnosis of HEV infection; we have shown that the disease is present in Egypt, that it can occur endemically as sporadic cases, and that children do have overt infection.The Lancet 03/1992; 339(8789):328-31. · 39.06 Impact Factor
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ABSTRACT: A newly developed assay for IgA class antibody to hepatitis E virus (IgA anti-HEV) was used to study 145 serum samples collected during an outbreak of an enterically transmitted hepatitis that occurred in 3 villages in the lower Shebeli region of Southern Somalia between January, 1988 and November, 1989. A total of 52.4% of the afflicted patients were found positive for IgA anti-HEV, and 73.1% of these were also positive for IgM. Both antibodies disappeared during the convalescence period. Similar results were also seen in serum obtained from sporadic cases of acute waterborne hepatitis in Pakistan.Journal of Medical Virology 09/1993; 40(4):334-8. · 2.37 Impact Factor