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.
Facultad de Biología, Centro de Estudios de la Proteínas, Universidad de la Habana, Ciudad Habana, Cuba. omarga@fbio.uh.cu
Omar A
OA
Gutiérrez
Emir
E
Salas
Yanko
Y
Hernández
Eduardo A
EA
Lissi
Gabriel
G
Castrillo
Osvaldo
O
Reyes
Hilda
H
Garay
Arístides
A
Aguilar
Beatriz
B
García
Anselmo
A
Otero
Maria A
MA
Chavez
Carlos A
CA
Duarte
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