Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay

  • Mark Page
  • Robin Thorpe
Part of the Springer Protocols Handbooks book series (SPH)


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.


Scintillation Counter Positive Antibody Soluble Antigen Packard Instrument Hemoglobin Solution 
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  1. 1.
    Johnstone, A. and Thorpe, R. (1996) Immunochemistry in Practice, 3rd ed. Blackwell Scientific, Oxford, UK.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Mark Page
    • 1
  • Robin Thorpe
    • 2
  1. 1.MEDEVA, Vaccine Research Unit, Department of BiochemistryImperial College of Science, Technology, and MedicineLondonUK
  2. 2.National Institute for Biological Standards and ControlPotters BarUK

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