Hybridoma Cultures for Production of Antibodies

Purification and Specificity of Antibodies
  • J. Denry Sato
Part of the Springer Protocols Handbooks book series (SPH)


During the process of characterizing the structure and function of novel antigens, it is usually necessary to create new monoclonal or polyclonal antibody reagents. However, once validated, these antibodies can be put to a multitude of experimental uses, such as detecting and quantitating antigens in cell and tissue extracts or biological fluids, purifying proteins for structural analyses, studying protein-protein interactions, and monitoring cellular differentiation. Although many well-characterized monoclonal antibodies (mAbs) to antigens of general interest are commercially available, mAbs to specialty antigens may need to be individually purified from hybridomas obtained from academic sources, or from not-for-profit cell repositories, such as the American Type Culture Collection ( This chapter describes protocols for producing, purifying, and verifying murine mAbs from pre-existing hybridomas.


Protein Antigen Column Volume Roller Bottle Murine IgG1 Murine mAbs 
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Further Reading

  1. Akerstrom, B., Brodin, T., Reis, K., and Bjorck, L. (1985), Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies. J. Immunol. 135, 2589–2592.PubMedGoogle Scholar
  2. Bruck, C, Portetelle, D., Glineur, C., and Bollen, A. (1982), One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel blue chromatography. J. Immunol. Methods 53, 313–319.PubMedCrossRefGoogle Scholar
  3. Ey, P. L., Prowse, S. J., and Jenkin, C. R. (1978), Isolation of pure IgG1, IgG2a, and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 15, 429–436.PubMedCrossRefGoogle Scholar
  4. Heide, K. and Schwick, H. G. (1978), Salt fractionation of immunoglobulins, in: Handbook of Experimental Immunology, vol. 1, 3rd ed., Weir, D. M., ed., Blackwell, Oxford, UK, pp. 7.1–7.11.Google Scholar
  5. Kawamoto, T., Sato, J. D., McClure, D. B., and Sato, G. H. (1986), Serum-free medium for the growth of NS-1 mouse myeloma cells and the isolation of NS-1 hybridomas. Methods Enzymol. 121, 266–277.PubMedCrossRefGoogle Scholar
  6. Myoken, Y., Okamoto, T., Osaki, T., Yabumoto, M., Sato, G. H., Takada, K., and Sato, J. D. (1989), An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells. In Vitro Cell Dev. Bìol. 25, 477–480.PubMedCrossRefGoogle Scholar
  7. Sato, J. D., Kawamoto, T., and Okamoto, T. (1987), Cholesterol requriement of P3-X63-Ag8 and X63-Ag8.653 myeloma cells for growth in vitro. J. Exp. Med. 165, 1761–1766.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • J. Denry Sato
    • 1
  1. 1.Division of Cell, Molecular, and Developmental BiologyAmerican Type Culture CollectionManassas

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