A drug discovery platform: A simplified immunoassay for analyzing HIV protease activity

• المؤلفون الرئيسيون: Kitidee K., Nangola S., Hadpech S., Laopajon W., Kasinrerk W., Tayapiwatana C.
• التنسيق: مقال
• اللغة: English
• منشور في: 2014
• الوصول للمادة أونلاين: http://www.scopus.com/inward/record.url?eid=2-s2.0-84869056201&partnerID=40&md5=e6e0f975bc764748d7d0494d567c6b8e; http://cmuir.cmu.ac.th/handle/6653943832/906

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 6MA-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. © 2012 Elsevier B.V.