Diagnosis of HIV-1 infection by detection of antibody IgG to HIV-1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens.
Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, 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 protein conjugate and recombinant protein-beta-D-galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of epsilon N-2,4-dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound beta-D-galactosidase activity by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers.
Hashida
S
S
Department of Biochemistry, Medical College of Miyazaki, Japan.
Hashinaka
K
K
Saitoh
A
A
Takamizawa
A
A
Shinagawa
H
H
Oka
S
S
Shimada
K
K
Hirota
K
K
Kohno
T
T
Ishikawa
S
S
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D015683:Gene Products, gag / Q000276:immunology; D015483:HIV Antibodies / Q000652:urine*; D015488:HIV Antigens / Q000276:immunology; D016655:HIV Core Protein p24 / Q000276:immunology; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000652:urine*; D012194:RNA-Directed DNA Polymerase / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015483:HIV Antibodies; D015488:HIV Antigens; D016655:HIV Core Protein p24; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase