ABSTRACT
Recombinant protein G was labelled with europium by conjugating the protein with Eu3+ chelate of a p-isothiocyanatobenzyl derivative of diethylenetriaminetetraacetic acid, a bifunctional chelating agent specifically optimized for labelling of immunoreagents with lanthanide ions. The labelling produced a universal reagent for time-resolved fluorometric immunoassays based on the principle of dissociative fluorescence enhancement (DELFIA). The optimum labelling level of about eight chelates per protein yielded a highly sensitive and stable reagent which retained its affinity for IgG and exhibited low non-specific binding to coated solid surfaces. The reagent was evaluated in an immunoassay of anti-tetanus antibodies in human serum samples and the results were compared to those obtained with Eu-labelled polyclonal and Eu-labelled monoclonal anti-human IgG antibodies. The detection limit of the assay was 0.003 mU/ml (0.3 microU per assay well). After a 100-fold dilution of the samples, the assay range extended from 0.3 mU/ml to 100,000 mU/ml with a linear range of five log orders. The incubation with Eu-labelled protein G reached equilibrium after a 15 min incubation. The rapid kinetics, the low non-specific background and the high specific binding suggest that Eu-protein G can serve as a universal label for immunoassays based on IgG binding to solid surfaces.
Subject(s)
Antibodies, Bacterial/analysis , Europium/chemistry , Immunoassay/methods , Immunoglobulin G/analysis , Nerve Tissue Proteins/chemistry , Humans , In Vitro Techniques , Kinetics , Protein Binding , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Tetanus Toxin/immunologyABSTRACT
Labelling of streptavidin with a fluorogenic europium ion was optimized with the aim of obtaining a universal, stable and highly sensitive non-isotopic label for time-resolved fluorometric immunoassays (TR-FIA) based on dissociative fluorescence enhancement (DELFIA). Even the conjugation of all the free amino groups of streptavidin with Eu chelates had only a minor effect on the binding capacity of the protein or its affinity. The labelled streptavidin was evaluated in indirect time-resolved immunofluorometric assays of human follicle and thyroid stimulating hormones (FSH and TSH). The interassay imprecision was below 3% within the concentration range from 2.5 to 94 U/l for the FSH samples and below 5% in the range from 2.4 to 35 mIU/l for the TSH samples. The detection limits of the assays for FSH and TSH were 0.05-0.10 U/l and 0.01-0.025 mIU/l, respectively, when a CV of 15% was regarded as the acceptable upper limit of imprecision. The results obtained by the indirect assays correlated closely with those obtained by corresponding direct sandwich assays. The model assays demonstrated the utility of Eu-labelled streptavidin as a universal reagent for immunoassays requiring a wide dynamic range and high sensitivity.
