Robert C. Morton

A new europium chelate for protein labelling and time-resolved fluorometric applications

Synthetic procedures are presented for a new chelator that forms stable and highly fluorescent complexes with Eu3+. This chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) is synthesized in a high-yield three-step procedure. BCPDA can be covalently incorporated into proteins under relatively mild conditions, and when complexed with Eu3+ forms a fluorescent product that has a lifetime in the range of 0.4 to 0.7 ms. Thus, it is useful for time-resolved fluorescence immunoassay applications.

Time-resolved fluorescence using a europium chelate of 4,7-bis-(chlorosulfopheny1)-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.

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.