Abstract
A large number of hybridoma culture supernatants (up to 200) need to be screened for antibodies at one time. The assay must be reliable so that it can accurately identify positive lines, and it must be relatively quick so that the positive lines, which are 75–100% confluent, can be fed and expanded as soon as possible after the assay results are known. Solid-phase binding assays are appropriate for this purpose and are commonly used for detection of antibodies directed against soluble antigens (1). The method involves immobilizing the antigen of choice onto a solid phase by electrostatic interaction between the protein and plastic support. Hybridoma supernatants are added to the solid phase (usually a 96-well format) in which positive antibodies bind to the antigen. Detection of the bound antibodies is then achieved by addition of an antimouse immunoglobulin labeled with radioactivity (usually 125I) and the radioactivity counted in a γ counter.
Similar content being viewed by others
Reference
Johnstone, A. and Thorpe, R. (1996) Immunochemistry in Practice, 3rd ed. Blackwell Scientific, Oxford, UK.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Page, M., Thorpe, R. (2009). Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_204
Download citation
DOI: https://doi.org/10.1007/978-1-59745-198-7_204
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-474-6
Online ISBN: 978-1-59745-198-7
eBook Packages: Springer Protocols