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

Abstract

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.

Advancing Laboratory Medicine through Innovation: A Tale of Six Inventors

“…When I joined, I took on the challenge of working with engineers, programmers, physicists, and others to perfect the instrument prototype. At the same time I initiated a program to optimize the reagents through novel conjugation techniques (3)(4)(5). In the end, we put together a combination of instrument/reagents, a product that was marketed successfully in Canada and abroad.…”

Sensitive determination of protein based on the fluorescence enhancement effect of terbium (III)–epinephrine–protein–sodium dodecylsulfate system

“…Recently, the rare earth ion complexes as fluorescence probes have been used for the determination of proteins. [11][12][13][14][15][16][17][18][19][20][21][22][23] However, the improvement of the sensitivities is limited because the fluorescence enhancement of rare earth ion complexes in these systems originates only from the hydrophobic microenvironment provided by protein. Therefore, it is imperative to find new rare earth ion complex as sensitive fluorescence probe for the determination of proteins.…”

Quantitative detection of cell surface protein expression by time‐resolved fluorimetry

“…It has been demonstrated in the literature that luminescence signals from long decay time lanthanide (Sm, Tb and Eu) and metalloporphyrin (Pd and Pt porphyrins) labels can be efficiently discriminated from each other and also from conventional prompt fluorescent labels by their characteristic excitation and emission wavelengths, narrow luminescence bandwidths, and luminescence decay times (5,15,(20)(21)(22)(23). In cell expression analysis, the multiple colour detection strategy would be valuable not only in increasing the number of expression targets (number of parameters) but, more importantly, to reduce signal variability originating from variation in the cell plating density.…”

“…In other words, the method does not allow collection of site-specific or morphological information, neither does it allow repeated and postponed expression analysis. In order to be applicable in imaging applications, and in postponed analysis, the DELFIA technique should be replaced by direct lanthanide fluorimetry (non-enhancement fluorimetry) that relies on stable fluorescent lanthanide chelates (23)(24)(25)(26). Such a stable chelate of Eu(III) was used in the present study, and these are also commercially available.…”