Development and Standardization of an Immuno-Quantified Solid Phase Assay for HIV-1 Aspartyl Protease Activity and Its Application to the Evaluation of Inhibitors

Publisher Website
Google Scholar
Abstract
The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.