Luminometric method for screening retroviral protease inhibitors.

Anal Biochem

Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 3, 166 28 Prague, Czech Republic.

Published: October 2005

We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.

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http://dx.doi.org/10.1016/j.ab.2005.07.013DOI Listing

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