Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin

A novel detection principle applicable for sensitive measurement of molecules of biological interest by time-resolved fluorescence spectrophotometry is described. Our method is based on the quantification of the Eu3+ chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in solution in presence of an excess of Eu3+ ions. BCPDA-labeled solid phase complexes obtained by conventional immunoassay procedures are transferred into solution using urea/SDS/Eu3+ as dissociating and fluorescent lanthanide ion reagent. Two 'sandwich-type' assay variants based on the above methodology were realized for the determination of small amounts of fibronectin (FN) in biological fluids. FN is captured from solution by solid phase coated gelatin or a monoclonal antibody, respectively. Rabbit anti-FN antiserum used as second antibody is detected with a biotinylated anti-rabbit IgG antibody. Fluoresence is measured after incubation with streptavidin-BCPDA and dissociation of solid phase complexes as described. Both assays have a detection limit (blank + 3 x SD) of less than 0.5 ng/ml FN, a dynamic range of up to 300 ng/ml, and intraserial coefficients of variation of 4.4 and 6.3%, respectively. Median FN concentrations in saliva of healthy individuals were 104 (gelatin) and 36 ng/ml (double antibody), respectively.