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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′

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′‐β‐D‐galactosidase conjugate was trapped onto polystyrene beads coated with affinity‐purified (anti‐2,4‐dinitrophenyl group) IgG, was eluted with ϵN‐2,4‐dinitrophenyl‐L‐lysine, and was transferred to polystyrene beads coated with streptavidin. β‐D‐Galactosidase activity bound to the streptavidin‐coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound β‐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 β‐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′‐β‐D‐galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at ‐20°C, and this increase was prevented without prolongation of the assay time for bound β‐D‐galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti‐p24 Fab′‐β‐D‐galactosidase conjugate for affinity‐purified rabbit anti‐p24 Fab′β‐D‐galactosidase conjugate. © 1996 Wiley‐Liss, Inc.