A sensitive time-resolved fluorescent immunoassay for metallothionein protein

Metallothioneins (MTs) are a family of low molecular weight metal-binding proteins induced by a broad range of stress conditions, including exposure to transition metal ions. Biochemical and immunological methods to measure NIT protein levels in tissues and cultured cells have been reported, but accuracy and sensitivity is impeded by high background levels, low specificity of currently available reagents, and relatively laborious and time-consuming multistep procedures. To address these difficulties, a protocol has been developed to measure MT protein levels using a competitive solid phase assay based on dissociation enhanced lanthanide fluoroimmuno (DELFIA) detection of ami-MT monoclonal antibody bound to solid phase MT. This assay allows time-resolved detection of antibody binding, based on binding and exchange of different lanthanide chelates followed by fluorescent detection, designed to reduce background fluorescence and increase sensitivity. The method allows measurement of low MT levels that are undetectable using current radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) protocols, and yields reproducible results with low background over a wide range of NIT concentrations. Improved sensitivity of NIT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function. (C) 2002 Elsevier Science B.V. All rights reserved.