Europium-labelled recombinant protein G. A fast and sensitive universal immunoreagent for time-resolved immunofluorometry.
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.
Markela
E
E
Wallac Oy, Turku, Finland.
Ståhlberg
T H
TH
Hemmilä
I
I
D000907:Antibodies, Bacterial / Q000032:analysis*; D005063:Europium / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D066298:In Vitro Techniques; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013744:Tetanus Toxin / Q000276:immunology
D000907:Antibodies, Bacterial; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D005063:Europium