The Genetic Systems HIV-1/HIV-2 PLUS O EIA is based on the principle of direct antibody sandwich technique. Microwell-strip plates (solid-phase) are coated with purified HIV antigens: gp190 and p24 recombinant proteins derived from HIV-1, gp36 peptide representing the immunodominant region of the HIV-2 transmembrane glycoprotein, and a synthetic polypeptide mimicking an artificial (not encoded by any existing virus) HIV-1 group O-specific epitope.
Serum samples and assay controls are added to the plate with specimen diluent containing a dye that changes color from purple to blue when combined with a specimen or control. The wells are incubated and then washed. Following addition of a colored conjugate solution (green) that contains the peroxidase-conjugated HIV-1 and HIV-2 antigens, the wells are incubated again. If HIV-1 or HIV-2 antibody is present, it will bind to the antigen coated on the well and to the peroxidase-conjugated antigens in the conjugate solution. The antigen-antibody-antigen complexes remain bound to the well during a subsequent wash step that will remove any unbound materials. Working chromogen solution (TMP) is added to the plate wells and allowed to incubate. A blue or blue-green color develops in proportion to the amount of HIV antibody present in the sample. Color development is stopped by the addition of acid, which changes the blue-green color to yellow. The optical absorbance of specimens and controls is determined spectrophotometrically at a wavelength of 450 nm.(Package insert: Genetics Systems HIV-1/HIV-2 PLUS O EIA. Bio-Rad Laboratories, Redman, WA)