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

Gutiérrez, Omar A.; Salas, Emir; Hernández, Yanko; Lissi, E. A.; Castrillo, Gabriel; Reyes, Osvaldo; Garay, Hilda; Aguilar, A.; García, Beatriz Fonseca; Otero, Anselmo; Chávez, María Á.; Duarte, Carlos A.

doi:10.1016/s0003-2697(02)00009-x

Cited by 23 publications

(26 citation statements)

References 18 publications

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“…The role of this cascade in many diseases has made it a primary target for many pharmaceuticals, including protease inhibitors. For the earlier reasons, there has been a proliferation of many protease assays, which include chromagenic substrates to assay proteolytic enzymes in blood coagulation pathways (16), ELISA (17), scintillation proximity (18), continuousflow approaches using FRET in solution-based fluorescence measurements to detect cleavage (19), cleavage of bioluminescent proteins linked to the surface of microtiter plates (20), and microtiter plate fluorescent assays using FRET-labeled substrates (21).…”

mentioning

confidence: 99%

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders¹,

Kim²,

Woods³

et al. 2006

Cytometry Pt A

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Background: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. Methods: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCytÒ flow cytometry system. Results: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition

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mentioning

confidence: 99%

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Saunders¹,

Kim²,

Woods³

et al. 2006

Cytometry Pt A

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“…Several related biochemical methods have been used to evaluate HIV-1 PI susceptibility ( Yu et al, 1995 ; Gutierrez et al, 2002 ; Hoffmann et al, 2005 ; Hu et al, 2005 ; Matsuda et al, 2007 ). The basic principle involved in these procedures is to incubate the recombinant PR, substrate peptide, and PI in vitro , and then measure the amount of substrate cleaved by PR.…”

Section: Discussionmentioning

confidence: 99%

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

et al. 2015

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Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

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“…There have been a number of studies that have considered putting surface enzyme kinetics into an algebraic framework that is usually based on a combination of Michaelis-Menten-type (noting that the enzyme is usually in excess of surface substrate rather than the other way around) and Langmuir-type kinetics (Gutiérrez et al 2002(Gutiérrez et al , 2005Lee et al 2006). Various models have been derived but these have been system-specifi c. For example, systems can involve either closed cells (Nayak et al 2007) or fl ow cells .…”

Section: The Kinetics Of Surface Enzymologymentioning

confidence: 99%

Detection of enzyme-catalyzed polysaccharide synthesis on surfaces

Clé

Martin

Field

et al. 2009

Biocatalysis and Biotransformation

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Strategically important cellular components, such as the cell wall and the starch granule, present surfaces during their biosynthesis and degradation. The enzymology of such surfaces is experimentally challenging and goes well beyond classical solution-state analyses. The kinetics of surface catalysis is complex but tractable. A number of approaches to monitor surface catalysis are reviewed and each is suited to a different biological problem. Particular attention is paid to a method we have recently developed for quantitatively monitoring polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy. This method has many attractive features with the potential to tackle both biological and industrial problems.

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

Cited by 23 publications

References 18 publications

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

Microsphere‐based protease assays and screening application for lethal factor and factor Xa

A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

Detection of enzyme-catalyzed polysaccharide synthesis on surfaces

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