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-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.

Development of a sensitive and specific xMAP assay for detection of antibodies against infectious laryngotracheitis and bronchitis viruses

“…MBMIs are typically biochemical tests that allow the detection or measurement of the concentration of a protein in a solution via an antibody or immunoglobulin [17]. MBMIs are often used in the diagnosis of various pathogens, such as human papillomavirus and human cytomegalovirus, to detect antibodies in serum samples [18][19][20]. The advantages of this technology are its simple operation, high accuracy, and reduction in the number This study describes the establishment and validation of single xMAP and duplex xMAP assays.…”

Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays

“…There is a strong trend towards the employment of multiplexed diagnostic methods in various clinical demands, such as detection of HLA antigens for transplants, chronic leukemias, microbiological diagnostic, and allergies as well as detection of hepatitis C virus [25, 28, 29]. The combination of microspheres and flow cytometry as a tool for diagnosis has not been solely used in the identification of human diseases.…”

“…These PE antibodies were chosen since they were used as reporter conjugates in multiplex assays in Luminex 100 ™ system (Luminex Corporation, Texas, USA). The conjugate best concentrations (0.5, 1, 2.5 and 5 μg/mL) or dilutions (1:3000, 1:1000 and 1:300) were determined according to the immunoassay protocol described elsewhere [28]. Stock solution concentrations of C and D conjugates, as obtained by spectrophotometry, were 1 mg/mL and 2.7 mg/mL respectively.…”

Immunodiagnostics and Immunosensor Design

“…The combined serological results often give reasonable information for the further treatment of a patient. Good examples are hepatitis C [194] and HIV diagnosis [195]. The methods to study disease subtypes and their association with therapeutic success have been improved.…”