title: Development of a multiplex bead-based assay for detection of hepatitis C virus.
abstract: Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.
authors.affiliation: Laboratório de Tecnologia Diagnóstica, CEP 21045-900 Rio de Janeiro, RJ, Brazil. bruna.fonseca@bio.fiocruz.br
authors.forename: Bruna P F
authors.initials: BP
authors.lastname: Fonseca
authors.forename: Christiane F S
authors.initials: CF
authors.lastname: Marques
authors.forename: Lílian D
authors.initials: LD
authors.lastname: Nascimento
authors.forename: Marcelle B
authors.initials: MB
authors.lastname: Mello
authors.forename: Leila B R
authors.initials: LB
authors.lastname: Silva
authors.forename: Nara M
authors.initials: NM
authors.lastname: Rubim
authors.forename: Leonardo
authors.initials: L
authors.lastname: Foti
authors.forename: Edimilson D
authors.initials: ED
authors.lastname: Silva
authors.forename: Antonio G P
authors.initials: AG
authors.lastname: Ferreira
authors.forename: Marco A
authors.initials: MA
authors.lastname: Krieger
mesh_terms: D000956:Antigens, Viral; D019411:Clinical Laboratory Techniques / Q000379:methods*; D016174:Hepacivirus / Q000276:immunology*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D018937:Hepatitis C Antibodies / Q000097:blood*; D006801:Humans; D008863:Microspheres; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D044967:Serum / Q000276:immunology; D014773:Virology / Q000379:methods*
chemical_list: D000956:Antigens, Viral; D018937:Hepatitis C Antibodies