Ultrasensitive immune complex transfer enzyme immunoassay of HIV-1 p24 antigen with less serum interference using 2,4-dinitrophenyl-anti-HIV-1 p24 IgG and indirectly immobilized (anti-2,4-dinitrophenyl group) Fab'

Abstract

In the immune complex transfer enzyme immunoassay previously reported, the immune complex consisting of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-HIV-1 p24 Fab' conjugate, HIV-1 p24 antigen and monoclonal mouse anti-HIV-1 p24 Fab'-β-D-galactosidase conjugate was trapped on polystyrene beads coated directly with affinity-purified (anti-2,4-dinitrophenyl group) IgG and was transferred to polystyrene beads coated with biotinyl-bovine serum albumin and streptavidin. The serum volume used was limited to 10 μL due to serious serum interference, and the detection limit of HIV-1 p24 antigen was 240 fg/mL serum. In the present study, HIV-1 p24 antigen was incubated simultaneously with 2,4-dinitrophenyl- affinity-purified rabbit anti-HIV-1 p24 IgG and monoclonal mouse anti-HIV-1 p24 Fab'-β-D-galactosidase conjugate in the presence of excess nonspecific rabbit IgG. The immune complex of the three components formed was trapped on polystyrene beads coated successively with biotinyl-bovine serum albumin, streptavidin and biotinyl-affinity-purified (anti-2,4-dinitrophenyl group) Fab'. After washing, the immune complex was eluted from the polystyrene beads with excess εN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads coated with affinity-purified goat (antirabbit IgG) IgG. The serum volume used was increased to 90 μL with only slight serum interference, and the detection limit of HIV-1 p24 antigen was lowered 9-fold to 26 fg/mL serum.