A solid-phase mutual inhibition assay with labeled antigen.

Abstract

A widely applicable method for the determination of the epitope specificities of a large number of monoclonal antibodies (MAbs) is presented. The method is based on the solid-phase mutual inhibition assay using 96-well plates coated with the respective MAbs, competitor MAbs, biotinylated antigen, and avidin-peroxidase conjugate. Using carcinoembryonic antigen (CEA) as a model antigen, the method was applied to determine epitope specificities of anti-CEA MAbs. A constant amount of biotinylated CEA was incubated with a given MAb immobilized on wells of 96-well plates in the presence of increasing amounts of soluble competitor MAbs. The biotinylated CEA bound to the immobilized antibody was then reacted with avidin-peroxidase conjugate and the activity of the bound peroxidase was determined by using o-phenylenediamine and hydrogen peroxide. The method used alleviates the laborious procedures of labeling all antibodies to be tested and the confusion caused by differential labeling among different MAbs. It is a convenient method for mapping analysis of many MAbs if the corresponding purified antigen is available.

• Authors: Masahide Kuroki
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 524 Pg. 59-66 ( 2009) ISSN: 1064-3745 [Print] United States
• PMID: 19377936 (Publication Type: Journal Article)

Chemical References

• Antibodies, Immobilized
• Antibodies, Monoclonal
• Carcinoembryonic Antigen
• Phenylenediamines
• avidin-horseradish peroxidase complex
• Avidin
• 1,2-diaminobenzene
• Hydrogen Peroxide
• Horseradish Peroxidase

Topics

• Animals
• Antibodies, Immobilized (analysis, chemistry, immunology)
• Antibodies, Monoclonal (analysis, chemistry, immunology)
• Avidin (metabolism)
• Binding, Competitive
• Biotinylation
• Carcinoembryonic Antigen (analysis, chemistry, immunology, metabolism)
• Epitope Mapping (economics, methods)
• Horseradish Peroxidase (metabolism)
• Humans
• Hydrogen Peroxide (metabolism)
• Isotope Labeling
• Phenylenediamines (metabolism)
• Radioimmunoassay