A Rapid Method for Generating Large Numbers of High-Affinity Monoclonal Antibodies from a Single Mouse

  • Nguyen thi Man
  • Glenn E. Morris
Part of the Springer Protocols Handbooks book series (SPH)


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, 3, 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, 6, 7, 8, 9, 10, 11, 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).


Creatine Kinase Myeloma Cell Microwell Plate Western Blotting Study Spleen Cell Suspension 
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Further Reading

  1. 1.
    Langone, J. J. and Van Vunakis, H. (eds.) (1986) Methods in enzymology, in Immunochemical Techniques, vol. 121, part I. Academic, New York.Google Scholar
  2. 2.
    Goding, J. W. (1986) Monoclonal Antibodies: Principles and Practice. Academic, New York.Google Scholar


  1. 1.
    Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.PubMedCrossRefGoogle Scholar
  2. 2.
    Galfre, G. and Milstein, C. (1981) Preparation of Monoclonal antibodies: strategies and procedures. Methods Enzymol. 73, 3–46.PubMedCrossRefGoogle Scholar
  3. 3.
    Fazekas de St. Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1–21.CrossRefGoogle Scholar
  4. 4.
    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.Google Scholar
  5. 5.
    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.CrossRefGoogle Scholar
  6. 6.
    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, 881,882PubMedCrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.CrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    Littlefield, J. W. (1964) Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709,710.PubMedCrossRefGoogle Scholar
  14. 14.
    Shulman, M., Wilde, C. D., and Kohler, G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269,270.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Nguyen thi Man
    • 1
  • Glenn E. Morris
    • 1
  1. 1.MRIC, NE Wales InstituteDeeside, ClwydUK

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