An anti-HIV p24 monoclonal coating antibody is adsorbed onto a microtiter plate. p24 antigen present in the sample or standard binds to the antibodies adsorbed on the plate; a FITC-conjugated mouse anti-p24 antibody is added and binds to p24 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-p24. 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 p24 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 HIV-1 p24 protein and sample p24 concentration is then determined.