Ultrasensitive and more specific enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV-1 in serum using affinity-purified rabbit anti-p24 Fab′ and monoclonal mouse anti-p24 Fab′

Abstract

Previously, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV-1 was developed. The immune complex comprising 2,4-dinitrophenyl-biotinyl-bovine serum albumin-rabbit anti-p24 Fab' conjugate, p24 antigen, and rabbit anti-p24 Fab' -beta-D-galactosidase conjugate was trapped onto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, was eluted with epsilon N-2, 4-dintrophenyl-L-lysine, and was transferred to polystyrene beads coated with streptavidin. beta-D-Galactosidase activity bound to the streptavidin-coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound beta-D-galactosidase activity had to be assayed for a long time (20 h), and the nonspecific signal was observed in 5% serum samples from subjects with low risk of HIV infection. In the present study, the assay time for bound beta-D-galactosidase activity was shortened to 2.5 h by using 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and affinity-purified rabbit anti-p24 Fab' -beta-D-galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at -20 degrees C, and this increase was prevented without prolongation of the assay time for bound beta-D-galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate for affinity-purified rabbit anti-p24 Fab'beta-D-galactosidase conjugate.