EUROPIUM-LABELED RECOMBINANT PROTEIN-G - A FAST AND SENSITIVE UNIVERSAL IMMUNOREAGENT FOR TIME-RESOLVED IMMUNOFLUOROMETRY

Recombinant protein G was labelled with europium by conjugating the pr otein with Eu3+ chelate of a p-isothiocyanatobenzyl derivative of diet hylenetriaminetetraacetic acid, a bifunctional chelating agent specifi cally optimized for labelling of immunoreagents with lanthanide ions. The labelling produced a universal reagent for time-resolved fluoromet ric immunoassays based on the principle of dissociative fluorescence e nhancement (DELFIA). The optimum labelling level of about eight chelat es per protein yielded a highly sensitive and stable reagent which ret ained its affinity for IgG and exhibited low non-specific binding to c oated solid surfaces. The reagent was evaluated in an immunoassay of a nti-tetanus antibodies in human serum samples and the results were com pared to those obtained with Eu-labelled polyclonal and Eu-labelled mo noclonal anti-human IgG antibodies. The detection limit of the assay w as 0.003 mU/ml (0.3 muU 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-labelle d protein G reached equilibrium after a 15 min incubation. The rapid k inetics, the low non-specific background and the high specific binding suggest that Eu-protein G can serve as a universal label for immunoas says based on IgG binding to solid surfaces.