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Development of a Multiplex Bead-Based Assay for Detection of Hepatitis C Virus
Bruna P. F. Fonseca,1* Christiane F. S. Marques,1 L¨ªlian D. Nascimento,1 Marcelle B. Mello,1
Leila B. R. Silva,1 Nara M. Rubim,1 Leonardo Foti,2,3 Edimilson D. Silva,1
Antonio G. P. Ferreira,1 and Marco A. Krieger2,3
Laborato´rio de Tecnologia Diagno´stica, Bio-Manguinhos-Fiocruz, Av. Brasil 4365, CEP 21040-360 Rio de Janeiro, RJ, Brazil1;
Instituto Carlos Chagas-Fiocruz, R. Professor Algacyr Munhoz Mader 3775, CEP 81350-010 Curitiba, PR, Brazil2; and
Instituto de Biologia Molecular do Parana´, R. Professor Algacyr Munhoz Mader 3775, CEP 81350-010 Curitiba, PR, Brazil3
Received 29 June 2010/Returned for modification 19 August 2010/Accepted 4 February 2011

Abstract£ºHepatitis 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-HCVpositive 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 timeeffectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.

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