Effect of Inducers, Incubation Time and Heme Concentration on IC₅₀ Value Variation in Anti-heme Crystallization Assay

Abstract

Heme detoxification through crystallization into hemozoin has been suggested as a good target for the development of screening assays for new antimalarials. However, comparisons among the data obtained from different experiments are difficult, and the IC₅₀ values (the concentrations of drug that are required to inhibit 50% of hemozoin formation) for the same drug vary widely. We studied the effects of changes in heme concentration (precursor of β-hematin), incubation time and three inducers (SDS, Tween 20 and linoleic acid) on the IC₅₀ of some antimalarials (chloroquine, quinine, amodiaquine, and clotrimazole). The results showed that increasing both inducer concentration and incubation time raised the IC₅₀ of selected antimalarials. Any change in those factors caused the IC₅₀ value to vary. Standardization of assay conditions is, therefore, necessary to increase reproducibility and reduce discrepancies in assay performance. Considering all of the variables, the best choice of inducers is in the order of SDS > Tween 20 > linoleic acid.