Time-resolved fluorescence using a europium chelate of 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labeling procedures and applications in immunoassays.
We describe optimal conditions for protein labeling with a new fluorescent probe, 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). The labeled proteins are suitable for time-resolved fluorometric applications because BCPDA forms fluorescent complexes with Eu3+ which exhibit very long fluorescence lifetimes. Labeling parameters such as the organic solvent used, pH, protein concentration, BCPDA excess and incubation times, were optimized accordingly. Excess BCPDA was removed by gel filtration and labeled proteins were characterized by absorbance and fluorescence measurements. The effect of labeling on the biological (binding) activity of the proteins streptavidin, avidin, monoclonal and polyclonal antibodies was also studied. It is shown that the labeled antibodies can be used for time-resolved fluoroimmunoassay applications.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal / Q000592:standards; D001360:Avidin; D001426:Bacterial Proteins; D002614:Chelating Agents; D005063:Europium; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000592:standards; D005456:Fluorescent Dyes; D006041:Goats; D006854:Hydrocortisone / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D010618:Phenanthrolines; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods / Q000592:standards; D019809:Streptavidin; D013997:Time Factors
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D010618:Phenanthrolines; C048844:anti-IgG; C054206:4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid; D001360:Avidin; D005063:Europium; D019809:Streptavidin; D006854:Hydrocortisone