Synthetic peptides representing immunodominant epitopes of HIV 1 and HIV 2 together with a monoclonal antibody to p24 HIV-1 antigen are coated onto wells of a microplate.
The peptides and the antibody have been carefully selected to ensure the screening of antibody and p24 antigen to all HIV-1 subtypes, including subtype O and HIV-2.
Serum or plasma samples are added to these wells and, if antibodies specific to HIV-1 and/or HIV-2 (IgG, IgM or IgA) are present in the sample, they will form stable complexes with the HIV peptide antigens in the well. In case HIV-1 p24 is present in the sample, the antigen will be captured by the specific monoclonal antibody.
Antigen-antibody complexes are then identified through the successive addition of:
- biotinylated peptides, a biotinylated monoclonal antibody to HIV-1 p24, and;
- horseradish peroxidase HRP Streptavidin conjugate.
The hydrolytic activity of horseradish peroxidase allows for the quantification of these antibody-antigen complexes. Peroxidase substrate solution is then added.
During incubation, a blue color will develop in proportion to the amount of anti-HIV-1/2 antibodies or HIV-1 p24 antigen bound to the well, thus establishing their presence or absence in the sample. Wells containing samples negative for anti-HIV antibody and/or p24 antigen remain colorless.
A stop solution is added to each well and the resulting yellow color is read on a microplate reader at 450 nm.