Protein Protocols Protein Protocols

162
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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Growth and Purification of Murine Monoclonal Antibodies
Mark Page and Robin Thorpe
1. Introduction
Once a hybridoma line has been selected and cloned, it can be expanded and seed
stocks cryopreserved for future use. Relatively large amounts of purified MAb may
also be required. There are a variety of procedures for this that ensure the establishment
of a stable cell line secreting high levels of specific immunoglobulin. High concentrations
of antibody can be generated by growing the line in the peritoneal cavity of mice/rats
of the same strain as the tumor cell line donor and spleen cell donor. Antibody is
secreted into the ascitic fluid formed within the cavity at a concentration up to 10 mg/mL.
However, the ascites will contain immunoglobulins derived from the recipient animal
that can be removed by affinity chromatography if desired. Several in vitro culture
methods using hollow fibres or dialysis tubing (1) have been developed and are commercially
available; this avoids the use of recipient mice/rats and contamination by
host immunoglobulins, although contamination with culture medium-derived proteins
may be a problem.
2. Materials
1. RPMI-1640/DMEM medium.
2. Fetal bovine serum (FBS).
3. Phosphate-buffered saline (PBS): 0.14 M NaCl, 2.7 mM KCl, 1.50 mM KH2PO4, 8.1 mM
Na2HPO4. 4. HT medium: 1 mL 50X HT supplement in 50 mL 10% FBS/RPMI.
5. HT supplement (Gibco, Paisley, UK).
3. Method
3.1. Growth of Hybridomas
1. After cloning, grow hybridomas in 1 mL HT medium in separate wells of a 24-well plate.
Check growth every day and feed with 0.5–1 mL HT medium if supernatant turns yellow
(see Note 1).
2. When cells are 75–100% confluent (see Note 2), expand into two further wells with fresh
HT medium. When these are confluent, transfer into a 25-cm3 flask and feed with
approx 10 mL medium (see Note 3). Subsequently, cell lines should be weaned off HT