Abstract
Since the first description by Kohler and Milstein (1), many variations on this method for the production of monoclonal antibodies (MAb) have appeared and recent comprehensive books on the subject have included both recom-binant and conventional methods (see Further Reading). The variation we describe here, however, includes a number of refinements that enable rapid (6–10 wk) production from a single spleen of 10–30 cloned and established hybridoma lines producing antibodies of high-affinity. We initially applied this method to recombinant fusion proteins containing fragments of the muscular dystrophy protein dystrophin (2–5), and dystrophin-related proteins (6,7), to hepatitis B surface antigen (8) and to the enzyme creatine kinase (CK) (9), and have used the MAb thus produced for immunodiagnosis, epitope mapping, and studies of protein structure and function (5–12). Epitopes shared with other proteins are common, so availability of several MAb against different epitopes on a protein can be important in ensuring the desired specificity in immunolo-calization and Western blotting studies (6). More recently, we have applied this same technique for nuclear proteins, such as emerin (16 MAbs; 13), lamin A (5 MAbs; 14), SMN (22 MAbs; 15), gemins (35 MAbs; 16) and the transcription factor, Six5 (18 MAbs; 17), as well as other human proteins as diverse as huntingtin (19 MAbs; 18) and dopamine receptors (8 MAbs; 19).
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References
Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.
Nguyen thi Man, Cartwright, A. J., Morris, G. E., Love, D. R., Bloomfield, J. F., and Davies, K. E. (1990) Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin. FEBS Lett. 262, 237–240.
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Pham, Y. C. N., Nguyen thi Man, Holt, I., Sewry, C. A., Pall, G., Johnson, K. J., and Morris, G. E. (2005) Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies. J. Cell. Biochem. 95, 990–1001.
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Shulman, M., Wilde, C. D., and Kohler, G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269–270.
Further Reading
Albitar, M. (ed.) (2007) Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology No 378) Humana Press, Totowa, NJ.
Shepherd, P. S. and Dean, C. (2000) Monoclonal Antibodies (A Practical Approach) Oxford University Press.
Morris, G. E. (ed.) (1996) Epitope Mapping Protocols (Methods in Mol. Biol. vol. 66) Humana Press, Totowa NJ.
Acknowledgment
We thank C. J. Chesterton (King’s College, London) for sharing with us his enthusiasm for, and experience of, hybridoma technology in 1981.
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Man, N.T., Morris, G.E. (2009). A Rapid Method for Generating Large Numbers of High-Affinity Monoclonal Antibodies from a Single Mouse. 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_208
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DOI: https://doi.org/10.1007/978-1-59745-198-7_208
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