Use of indirectly immobilized recombinant p17 antigen for detection of antibodies to HIV-1 by enzyme immunoassay.

Recombinant HIV-1 p17 antigen (rp17) and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption, and biotinyl-rp17, biotinyl-MBP-rp17, and 2,4-dinitrophenyl (DNP)-MBP-rp17 were immobilized indirectly onto polystyrene beads, which had been coated with streptavidin alone, with biotinyl-bovine serum albumin and streptavidin and with (anti-2,4-dinitrophenyl group) IgG. These immobilized antigens were tested by incubation with diluted serum from an HIV-1 seropositive subject in the absence and presence of serum from HIV-1 seronegative subjects and, after washing, with rp17 beta-D-galactosidase conjugate. Higher positive signals (fluorescence intensities for bound -beta-D-galactosidase activity) and less serum interference were obtained with indirectly immobilized antigens than with directly immobilized ones. Enzyme immunoassay using biotinyl-MBP-rp17 indirectly immobilized onto polystyrene beads, which had been coated sequentially with biotinyl-bovine serum albumin and streptavidin, was approximately 1,000-fold more sensitive than that using directly immobilized rp17 antigen and Western blotting for p17 band. This enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as or even earlier than conventional methods and considerably earlier than Western blotting for HIV-1 p17 band. In addition, the sensitivity was further improved approximately 10-fold by incubation with shaking for immunoreactions and by increase of both the number of polystyrene beads and the volume of serum samples used per assay.