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 MT 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 anti-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 MT concentrations. Improved sensitivity of MT protein detection is of value in toxicological measurement of stress responses and assessment of MT expression and function.
Butcher
Heather
H
London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6.
Kennette
Wendy
W
Collins
Olga
O
Demoor
Janice
J
Koropatnick
James
J
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D015200:Fluoroimmunoassay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D028581:Lanthanoid Series Elements; D008668:Metallothionein / Q000032:analysis* / Q000276:immunology; D051379:Mice; D012015:Reference Standards; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D028581:Lanthanoid Series Elements; D008668:Metallothionein