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

Overview

Journal J Clin Lab Anal

Publisher Wiley

Specialties Biomedical Engineering
Biotechnology
Pathology

Date 1999 May 14

PMID 10323478

Authors

S Ishikawa

S Hashida

K Hashinaka

A Saito

A Takamizawa

H Shinagawa

E Ishikawa

Affiliations

Soon will be listed here.

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'-beta-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 microL 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'-beta-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 epsilonN-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 microL with only slight serum interference, and the detection limit of HIV-1 p24 antigen was lowered 9-fold to 26 fg/mL serum.

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