Determination of epitope specificities and affinities of monoclonal antibodies in solution phase using biotin-labeled carcinoembryonic antigen and avidin as precipitating agent.
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.
Krüger
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U
Institute of Clinical Chemistry and Pathobiochemistry, University of Technology, Aachen, F.R.G.
Wickert
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L
Wagener
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C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000915:Antibody Affinity; D001360:Avidin; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001710:Biotin; D002272:Carcinoembryonic Antigen / Q000276:immunology; D000939:Epitopes / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis; D007700:Kinetics; D011233:Precipitin Tests / Q000379:methods; D011863:Radioimmunoassay / Q000379:methods; D012996:Solutions
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D012996:Solutions; D001360:Avidin; D001710:Biotin