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For diagnosis of HIV-1 infection, attempts were made to detect anti-HIV-1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT) and p17 as antigens. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli beta-D-galactosidase for p17. 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. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV-1 infection possible.

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.

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