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

Abstract

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 β‐d‐galactosidase conjugate. Higher positive signals (fluorescence intensities for bound ‐β‐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. J. Clin. Lab. Anal. 13:9–18, 1999. © 1999 Wiley‐Liss, Inc.