Protein Protocols Protein Protocols

Making Bispecific Antibodies 1115
160
Screening Hybridoma Culture Supernatants
Using Solid-Phase Radiobinding Assay
Mark Page and Robin Thorpe
1. Introduction
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 125 I) and the radioactivity counted in a γ counter.
2. Materials
1. Antigen: 0.5–5 µg/mL in phosphate-buffered saline (PBS) with 0.02% sodium azide.
2. PBS: 0.14 M NaCl, 2.7 mM KCl, 1.50 mM KH2PO4, 8.1 mM Na2HPO4. 3. Blocking buffer: PBS containing either 5% v/v pig serum, 5% w/v dried milk powder, or
3% w/v hemoglobin (see Note 1).
4. Hybridoma culture supernatants.
5. Negative control: irrelevant supernatant or culture medium.
6. Positive control: serum from immunized mouse from which spleen for hybridoma production
was derived.
7. Antimouse/rat immunoglobulin, 125I-labeled (100 µCi/5 µg protein).
3. Method
1. Pipet 50 µL of antigen solution into each well of a 96-well microtiter plate and incubate at
4°C overnight. Such plates can be stored at 4°C for several weeks. Seal plates with cling
film to prevent the plates from drying out (see Note 2).
2. Remove antigen solution from wells by either a Pasteur pipet (cover the tip with a small
piece of plastic tubing to prevent scratching the antigen-coated wells) or by shaking the
contents from the plate in one quick movement (see Note 3).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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