Immunoassays for pentamidine and related compounds: Development of a facile inhibitory ELISA suitable for clinical use

Aromatic dicationic drugs have a broad spectrum of activity against protozo al and fungal pathogens including Pneumocystis carinii, Leishmania mexicana amazonensis, Cryptosporidium parvum and Cryptococcus neoformans. Pentamidi ne serves as the exemplar for an extensive collection of newly synthesized related compounds, which have reduced toxicity and a wider range of target organisms. Assays of pentamidine and related compounds have depended on HPL C-tandem mass spectrometry (HPLC-TMS) for the quantitation and identificati on of drug and metabolites. Immunoassays for pentamidine would have many ad vantages over the HPLC methods including relative simplicity of assay forma t and required equipment, convenience in sample preparation and reduction i n time and cost of assays. In this report we describe a simple ELISA based immunoassay for pentamidine and pentamidine-like drugs with requisite sensitivity and specificity for use as a clinical assay (EC50 value of about 50 nanomolar). Immunogen was s ynthesized by coupling the hapten aminopentamidine to ovalbumin (chemically modified to provide an optimal number of SH groups) using sulfo-MBS. Malei c-anhydride activated ELISA plates were covalently sensitized using the ami nopentamidine hapten and used in an inhibitory ELISA assay format whereby t he ability of analyte to suppress antibody binding to sensitized plate was measured. The assay detects primarily the phenolic amidine of pentamidine w hen in a para position and hence can also detect structurally related deriv atives of pentamidine of potential interest as new therapeutic agents. J. C lin. Lab. Anal. 14:73-82, 2000. (C) 2000 Wiley-Liss, Inc.