Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays

Multiple fluorescence labeling with conventional probes like fluorescein, to improve the detection limit of labeled reactants, is not usually successful because of fluorescence quenching. In contrast, we found that the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) can be incorporated into proteins at very high molar ratios. Working with thyroglobulin as a model protein, we found that when 160 BCPDA molecules are incorporated into one thyroglobulin molecule, the fluorescence emitted by the labeled protein in the presence of excess Eu3+, is equivalent to that emitted by approximately 900 molecules of unconjugated BCPDA:Eu3+ complexes. We took advantage of the lack of any quenching effects and of the enhancement observed with the multiply labeled protein, to develop a universal reagent system consisting of (a) streptavidin covalently coupled to BCPDA labeled thyroglobulin and (b) excess Eu3+. With this approach, streptavidin is heavily labeled through thyroglobulin and retains its full biotin binding activity. We used the reagent to develop a highly sensitive time-resolved heterogeneous immunofluorometric assay of alpha-fetoprotein (AFP) in serum, using monoclonal antibodies. One antibody is immobilized in white microtitration wells (solid-phase) and the other is biotinylated. We demonstrate that this assay, using the newly developed reagent, is 25-fold more sensitive than the one using directly BCPDA labeled antibody and 5-fold more sensitive than an assay that uses BCPDA-labeled streptavidin. The detection limit of the assay with the new reagent was down to 60 amol of AFP per well. We conclude that multiple fluorescence labeling with europium chelators is an effective method of extending the sensitivity of currently used fluorescence immunoassay procedures.