Abstract
Since the first description by Kohler and Milstein (1), many variations on this method for the production of monoclonal antibodies (MAb) have appeared (e.g., see refs. 2–4 and Chapter 143), and it may seem superfluous to add another. The variation we describe here, however, includes a number of refinements that enable rapid (6–10 wk) production from a single spleen of large numbers (20–30) of cloned, established hybridoma lines producing antibodies of high-affinity. We have applied this method to recombinant fusion proteins containing fragments of the muscular dystrophy protein dystrophin (5,6), and dystrophin-related proteins (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 immunolocalization and Western blotting studies (7).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Further Reading
Langone, J. J. and Van Vunakis, H. (eds.) (1986) Methods in enzymology, in Immunochemical Techniques, vol. 121, part I. Academic, New York.
Goding, J. W. (1986) Monoclonal Antibodies: Principles and Practice. Academic, New York.
References
Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.
Galfre, G. and Milstein, C. (1981) Preparation of Monoclonal antibodies: strategies and procedures. Methods Enzymol. 73, 3–46.
Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1–21.
Zola, H. and Brooks, D. (1982) Techniques for the production and characterization of monoclonal hybridoma antibodies, in Monoclonal Antibodies: Techniques and Applications (Hurrell, J. G., ed.), CRC, FL, pp. 1–57.
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.
Ellis, J. M., Nguyen thi Man, Morris, G. E., Ginjaar, I. B., Moorman, A. F. M., and van Ommen, G.-J. B. (1990) Specifity of dystrophin analysis improved with monoclonal antibodies. Lancet 336, 881,882
Nguyen thi Man, Ellis, J. M., Love, D. R., Davies, K. E., Gatter, K. C., Dickson, G., and Morris, G. E. (1991) Localization of the DMDL-gene-encoded dystrophin-related protein using a panel of 19 monoclonal antibodies. Presence at neuromuscular junctions, in the sarcolemma of dystrophic skeletal muscle, in vascular and other smooth muscles and in proliferating brain cell lines. J. Cell Biol. 115, 1695–1700.
Le Thiet Thanh, Nguyen thi Man, Buu Mat Phan, Ngoc Tran Nguyen, thi Vinh Ha, and Morris, G. E. (1991) Structural relationships between hepatitis B surface antigen in human plasma and dimers of recombinant vaccine: a monoclonal antibody study. Virus Res. 21, 141–154.
Nguyen thi Man, Cartwright, A. J., Andrews, K. M., and Morris, G. E. (1989) Treatment of human muscle creatine kinase with glutaraldehyde preferentially increases the immunogenicity of the native conformation and permits production of high-affinity monoclonal antibodies which recognize two distinct surface epitopes. J. Immunol. Methods 125, 251–259.
Nguyen thi Man, Cartwright, A. J., Osborne, M., and Morris, G. E. (1991) Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies. Biochim. Biophys. Acta 1076, 245–251.
Morris, G. E. and Cartwright, A. J. (1990) Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase. Biochim. Biophys. Acta 1039, 318–322.
Sedgwick, S. G., Nguyen thi Man, Ellis, J. M., Crowne, H., and Morris, G. E., (1991) Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Research 19, 5889–5894.
Littlefield, J. W. (1964) Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709,710.
Shulman, M., Wilde, C. D., and Kohler, G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269,270.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Man, N.t., Morris, G.E. (1996). 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. https://doi.org/10.1007/978-1-60327-259-9_144
Download citation
DOI: https://doi.org/10.1007/978-1-60327-259-9_144
Publisher Name: Humana Press
Print ISBN: 978-0-89603-338-2
Online ISBN: 978-1-60327-259-9
eBook Packages: Springer Book Archive