title: A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.
abstract: Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.
authors: Division of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, 50200, Thailand.
authors: Kuntida
authors: K
authors: Kitidee
authors: Sawitree
authors: S
authors: Nangola
authors: Sudarat
authors: S
authors: Hadpech
authors: Witida
authors: W
authors: Laopajon
authors: Watchara
authors: W
authors: Kasinrerk
authors: Chatchai
authors: C
authors: Tayapiwatana
mesh_terms: D000911:Antibodies, Monoclonal; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006678:HIV / Q000187:drug effects* / Q000201:enzymology*; D015483:HIV Antibodies; D016333:HIV Protease / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000302:isolation & purification*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D017320:HIV Protease Inhibitors; D016333:HIV Protease