An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity.
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.
Gutiérrez
Omar A
OA
Facultad de Biología, Centro de Estudios de la Proteínas, Universidad de la Habana, Ciudad Habana, Cuba. omarga@fbio.uh.cu
Salas
Emir
E
Hernández
Yanko
Y
Lissi
Eduardo A
EA
Castrillo
Gabriel
G
Reyes
Osvaldo
O
Garay
Hilda
H
Aguilar
Arístides
A
García
Beatriz
B
Otero
Anselmo
A
Chavez
Maria A
MA
Duarte
Carlos A
CA
D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D015483:HIV Antibodies / Q000276:immunology; D016333:HIV Protease / Q000032:analysis*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007124:Immunoenzyme Techniques
D015483:HIV Antibodies; D017320:HIV Protease Inhibitors; D016333:HIV Protease