An anti-HCVcAg monoclonal coating antibody is adsorbed onto a microtiter plate. HCV core antigen present in the sample or standard binds to the antibodies adsorbed on the plate; a FITC-conjugated mouse anti-HCVcAg antibody is added and binds to the antigen captured by the first antibody.
Following incubation and wash steps, a HRP-conjugated mouse anti-FITC antibody is added and binds to the FITC conjugated anti-HCVcAg. Following unbound HRP-conjugated mouse anti-FITC antibody is removed during a wash step, and substrate solution reactive with HRP is added to the wells.
A colored product is formed in proportion to the amount of HCV core antigen present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from recombinant HCV core antigen and sample concentration is then determined.