Determination of epitope specificities and affinities of monoclonal antibodies in solution phase using biotin-labeled carcinoembryonic antigen and avidin as precipitating agent

J Immunol Methods. 1989 Feb 8;117(1):25-32. doi: 10.1016/0022-1759(89)90114-2.

Abstract

A generally applicable method for the selective precipitation of antigens from solution phase is presented. The method is based on the rapid and quantitative binding of avidin to biotin-labeled antigen in the presence of biotinylated carrier protein. Using carcinoembryonic antigen (CEA) as a model antigen biotin-labeled CEA was precipitated to greater than 97% at amounts of up to 5000 ng/tube. The method was applied to the determination of epitope specificities and affinities of monoclonal anti-CEA antibodies in solution phase. Non-specific precipitation did not exceed 2% for radiolabeled Fab fragments and 6% for radiolabeled IgG. Because antigen-antibody binding takes place in solution, artifacts introduced by the immobilization of reactants to solid supports are omitted.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / analysis*
  • Antibody Affinity*
  • Avidin*
  • Binding Sites, Antibody
  • Binding, Competitive
  • Biotin*
  • Carcinoembryonic Antigen* / immunology
  • Epitopes / immunology*
  • Immunoglobulin Fab Fragments / analysis
  • Kinetics
  • Precipitin Tests / methods
  • Radioimmunoassay / methods
  • Solutions

Substances

  • Antibodies, Monoclonal
  • Carcinoembryonic Antigen
  • Epitopes
  • Immunoglobulin Fab Fragments
  • Solutions
  • Avidin
  • Biotin