Protein Protocols Protein Protocols

161
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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Screening Hybridoma Culture Supernatants Using ELISA
Mark Page and Robin Thorpe
1. Introduction
Enzyme-linked immunosorbent assay (ELISA) is a widely used method for the detection
of antibody and is appropriate for use for screening hybridoma supernatants (1).
As with radiobinding assays, it is a solid-phase binding assay that is quick, reliable, and
accurate. The method is often preferred to radioactive assays, since the handling and
disposal of radioisotopes is avoided and the enzyme conjugates are more stable than
radioiodinated proteins. However, most of the substrates for the enzyme reactions are
carcinogenic or toxic and, hence, require handling with care.
Enzymes are selected that show simple kinetics, and can be assayed by a simple
procedure (normally spectrophotometric). Cheapness, availability, and stability of substrate
are also important considerations. For these reasons, the most commonly used
enzymes are alkaline phosphatase, β-D-galactosidase, and horseradish peroxidase.
2. Materials
1. PBS-Tween: Phosphate-buffered saline (PBS) (0.14 M NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4, 8.1 mM Na2HPO4) containing 0.05% Tween-20.
2. Antigen: 1–5 µg/mL in PBS.
3. Blocking buffers: PBS-Tween containing either 5% v/v pig serum or 5% w/v dried milk
powder (see Note 1).
4. Hybridoma culture supernatants.
5. Alkaline phosphatase conjugated antimouse immunoglobulin.
6. Carbonate buffer: 0.05 M sodium carbonate, pH 9.6.
7. p-Nitrophenyl phosphate, disodium hexahydrate: 1 mg/mL in carbonate buffer containing
0.5 mM magnesium chloride.
8. 1 M sodium hydroxide.
3. Method
1. Pipet 50 µL of antigen solution into each well of a 96-well plate, and incubate overnight at
4°C. Such plates can be stored for 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).