Europium-labelled recombinant protein G. A fast and sensitive universal immunoreagent for time-resolved immunofluorometry

J Immunol Methods. 1993 May 5;161(1):1-6. doi: 10.1016/0022-1759(93)90192-a.

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.

MeSH terms

  • Antibodies, Bacterial / analysis*
  • Europium / chemistry*
  • Humans
  • Immunoassay / methods*
  • Immunoglobulin G / analysis*
  • In Vitro Techniques
  • Kinetics
  • Nerve Tissue Proteins / chemistry*
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Spectrometry, Fluorescence
  • Tetanus Toxin / immunology

Substances

  • Antibodies, Bacterial
  • G-substrate
  • Immunoglobulin G
  • Nerve Tissue Proteins
  • Recombinant Proteins
  • Tetanus Toxin
  • Europium