Immune complex transfer enzyme immunoassay that is more sensitive and specific than western blotting for detection of antibody immunoglobulin G to human immunodeficiency virus type 1 in serum with recombinant pol and gag proteins as antigens.
Antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) in serum was detected by ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) with recombinant reverse transcriptase (rRT), p17 (rp17) and p24 (rp24) of HIV-1 as antigens and beta-D-galactosidase from Escherichia coli as the label. The immune complex, comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate, antibody IgG to HIV-1, and recombinant protein-beta-D-galactosidase conjugate, was trapped on polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene beads coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. The assays were highly reproducible with no serious serum interference, and they were much more sensitive than Western immunoblotting for the corresponding antigens. Signals with rRT, rp17, and rp24 for asymptomatic carriers were at least 56,000-, 680-, and 22-fold higher, respectively, than those for seronegative individuals, and neither indeterminate nor false-positive results were observed, whereas some serum samples were false negative or false positive by Western blotting for p17 and/or p24 antigen. In some cases, seroconversion was detected earlier than by conventional methods. Therefore, these assays are suggested to be more useful than conventional methods not only for the confirmation of antibody IgGs to RT, p17, and p24 of HIV-1 in serum but also for the detection of seroconversion.
Department of Biochemistry, Miyazaki Medical College, Japan.
S
S
Hashida
K
K
Hashinaka
I
I
Nishikata
S
S
Oka
K
K
Shimada
A
A
Saito
A
A
Takamizawa
H
H
Shinagawa
S
S
Yano
H
H
Kojima
D000936:Antigen-Antibody Complex / Q000097:blood / Q000737:chemistry; D015153:Blotting, Western; D015683:Gene Products, gag / Q000276:immunology*; D015684:Gene Products, pol / Q000276:immunology*; D015483:HIV Antibodies / Q000032:analysis / Q000276:immunology*; D015488:HIV Antigens / Q000276:immunology*; D006679:HIV Seropositivity / Q000097:blood / Q000175:diagnosis; D006801:Humans; D007124:Immunoenzyme Techniques / Q000592:standards; D007074:Immunoglobulin G / Q000032:analysis; D012680:Sensitivity and Specificity
D000936:Antigen-Antibody Complex; D015683:Gene Products, gag; D015684:Gene Products, pol; D015483:HIV Antibodies; D015488:HIV Antigens; D007074:Immunoglobulin G