Test ID: HCVPS    
Hepatitis C Antibody Screen Patient Source, Serum

Chemiluminescent Immunoassay (CIA):  

The VITROS anti-HCV assay is performed using the VITROS Anti-HCV Reagent Pack and VITROS Immunodiagnostic Products Anti-HCV Calibrator on the VITROS ECi Immunodiagnostic System (Ortho-Clinical Diagnostics, Inc., Raritan, NJ). An immunometric technique is used, involving a 2-stage reaction. In the first stage, hepatitis C virus (HCV) antibody present in the sample binds to HCV recombinant antigens coated on the reaction wells, and unbound sample is removed by washing. In the second stage, horseradish peroxidase (HRP)-labeled antibody conjugate (mouse monoclonal antihuman IgG) binds to human IgG captured on the well in the first stage. Unbound conjugate is removed by washing. A reagent containing luminogenic substrates (a luminal derivative and a peracid salt) and an electron transfer agent is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminal derivative, producing light. The electron transfer agent increases the level and duration of the light produced. The emitted light signals are detected and measured by the VITROS ECi Immunodiagnostic System. The amount of HRP conjugate bound is directly proportional to the level of anti-HCV antibodies present in a given sample. (Ismail N, Fish GE, Smith MN: Laboratory evaluation of a fully automated chemiluminescence immunoassay for rapid detection of HBsAg, antibodies to HBsAg, and antibodies to hepatitis C virus. J Clin Microbiol 2004;42:610-617)

RT-PCR:

The COBAS AmpliPrep/COBAS TaqMan HCV Test is a Food and Drug Administration (FDA)-approved in vitro assay based on an automated method (AmpliPrep) for nucleic acid extraction and purification and a homogeneous (real-time) PCR method (TaqMan) for amplifying the HCV-specific 5' UTR sequence (241 bp size) and generating a signal by cleaving a target-specific TaqMan probe during amplification. The probe contains a reporter fluorophore and a quencher dye that absorbs light emitted by the reporter. Cleavage of the probe physically separates the quencher from the reporter, enabling light emitted by the latter to be detected by a photomultiplier tube. Because amplification and detection are performed simultaneously, amplification products are measured during the exponential phase of DNA amplification regardless of the initial target concentration. (Package insert: COBAS AmpliPrep/COBAS TaqMan HCV Test; Roche Molecular Systems, Inc., Branchburg, NJ)