Summary
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.
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References
Kaufman, B. M., and Goldsby, R. A. (1982) Epitope ratio analysis (ERA): a simple radioimmunological method using monoclonal antibodies for the simultaneous analysis of several antigens. J. Immunol. Methods 54, 1–7.
Wagener, C., Yang, Y. H. J., Crawford, F. G., and Shively, J. E. (1983) Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies. J. Immunol. 130, 2308–2315.
Bayer, E. A., and Wilchek, M. (1990) Protein biotinylation. Methods Enzymol. 184, 138–160.
Harlow, E., and Lane, D. (1989) Antibodies: A Laboratory Manual, 2nd ed., Cold Spring Harbor Lab., Cold Spring Harbor, NY.
Kuroki, M., Wakisaka, M., Murakami, M., Haruno, M., Arakawa, F., Higuchi, H., and Matsuoka, Y. (1992) Determination of epitope specificities of a large number of monoclonal antibodies by solid-phase mutual inhibition assays using biotinylated antigen. Immunol. Invest. 21, 523–538.
Kuroki, M., Greiner, J. W., Simpson, J. W., Primus, F. J., Guadagni, F., and Schlom, J. (1989) Serologic mapping and biochemical characterization of the carcinoembryonic antigen epitopes using fourteen monoclonal antibodies. Int. J. Cancer 44, 208–218.
Kuroki, M., Arakawa, F., Haruno, M., Murakami, M., Wakisaka, M., Higuchi, H., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1992) Biochemical characterization of 25 distinct carcinoembryonic antigen (CEA) epitopes recognized by 57 monoclonal antibodies and categorized into 7 groups in terms of domain structure of the CEA molecule. Hybridoma 11, 391–407.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.
Savage, M. D., Mattson, G., Desai, S., Nielander, G. W., Morgensen, S., and Conklin, E. J. (1994) Avidin–Biotin Chemistry: A Handbook, 2nd ed., Pierce Chemical Co., Rockford, IL.
Wilchek, M., and Bayer, E. A. (1990) Introduction to avidin–biotin technology. Methods Enzymol. 184, 5–13.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Kuroki, M. (2009). A Solid-Phase Mutual Inhibition Assay with Labeled Antigen. In: Schutkowski, M., Reineke, U. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 524. Humana Press. https://doi.org/10.1007/978-1-59745-450-6_4
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DOI: https://doi.org/10.1007/978-1-59745-450-6_4
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