Detection of competitive enzyme inhibition with end point progress curve data
2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 358, no 1, 11-19 p.Article in journal (Refereed) Published
A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.
Place, publisher, year, edition, pages
2006. Vol. 358, no 1, 11-19 p.
Limit of detection, Enzyme inhibition, High-throughput screening, Primary screening, Robustness, Z′ factor, Enzymatic assays, Reaction progress curves, Error structure
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:uu:diva-94505DOI: 10.1016/j.ab.2006.07.008ISI: 000241428000002PubMedID: 16978578OAI: oai:DiVA.org:uu-94505DiVA: diva2:168375