Detection of antibody IgG to HIV‐1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV‐1 infection

Abstract

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.