Pentamer Formation Assay Based on Drug Functional Mechanisms
In performing the Pentamer Formation Assay (PFA), as in the case for other immunoassays, extracts of blood containing tacrolimus or cyclosporin A (CsA) should be free of endogenous immunophilins which, if present, might interfere with the assay. The adverse events directly associated with tacrolimus should be reversible to the cells, and irreversible damage is considered to be through indirect mechanism. Enzyme-linked immunosorbent assay (ELISA)-based method is available for tacrolimus and for CsA. Calcineurin is a serine/threonine phosphatase that acts downstream in T-cell receptor signal transduction pathways. The PFA is a mechanism-based assay for immunosuppressant tacrolimus and CsA. As shown by tacrolimus and CsA metabolites, receptor binding does not always correlate with and predict their immunosuppressive activity. Endogenous immunophilins from the patient sample would be also subject to competitive binding of the drug(s), with the exogenous assay immunophilins for complex formation.