Portal de Periódicos da CAPES

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity

2002; Elsevier BV; Volume: 307; Issue: 1 Linguagem: Inglês

10.1016/s0003-2697(02)00009-x

1096-0309

Omar A. Gutiérrez, Emir Salas‐Sarduy, Yanko Hernández, E. A. Lissi, Gabriel Castrillo, Osvaldo Reyes, Hilda Garay, Arı́stides Aguilar, Beatriz Garcı́a, Anselmo Otero, María Á. Chávez, Carlos A. Duarte,

HIV-related health complications and treatments

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.