More sensitive immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1 with shorter incubation time for immunoreactions and larger volumes of serum samples.
In the previous immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1, the immune complex comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 conjugate, anti-p17 IgG, and recombinant p17-beta-D-galactosidase conjugate was trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by overnight incubation and was transferred to polystyrene beads coated with (antithuman IgG gamma-chain) IgG by 3 hr incubation in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine. These processes were made efficient by incubation with shaking and by using solid phases with larger surface areas. In addition, the volume of serum samples used was increased from 10 microliters to 100 microliters. As a result, the sensitivity was improved 20-30-fold and was approximately 100,000-fold higher than that of Western blotting for p17 band, even when both trapping and transferring of the immune complex were performed for only 30 min. Furthermore, testing many samples became easily possible with higher sensitivity using microplates and a fluororeader.
Ishikawa
S
S
Department of Biochemistry, Miyazaki Medical College, Japan.
Hashida
S
S
Hashinaka
K
K
Kojima
M
M
Saito
A
A
Takamizawa
A
A
Shinagawa
H
H
Oka
S
S
Shimada
K
K
Ishikawa
E
E
D019297:2,4-Dinitrophenol; D000328:Adult; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D005260:Female; D015683:Gene Products, gag / Q000276:immunology*; D015483:HIV Antibodies / Q000097:blood*; D015488:HIV Antigens / Q000276:immunology*; D006679:HIV Seropositivity / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000097:blood*; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D011137:Polystyrenes; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins; D001616:beta-Galactosidase; D054301:gag Gene Products, Human Immunodeficiency Virus
D000936:Antigen-Antibody Complex; D015683:Gene Products, gag; D015483:HIV Antibodies; D015488:HIV Antigens; D007074:Immunoglobulin G; D011137:Polystyrenes; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D012710:Serum Albumin, Bovine; D001616:beta-Galactosidase; D019297:2,4-Dinitrophenol