Production of Monoclonal Antibodies

Wayne M. Yokoyama1

1 University of California School of Medicine, San Francisco, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Appendix 3J
DOI:  10.1002/0471142956.cya03js37
Online Posting Date:  August, 2006
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Abstract

This unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium (thymocyte‚Äźconditioned medium).

     
 
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Table of Contents

  • Basic Protocol 1: Immunization to Produce Monoclonal Antibodies
  • Basic Protocol 2: Cell Fusion and Selection of Hybridomas
  • Support Protocol 1: Screening Primary Hybridoma Supernatants
  • Support Protocol 2: Establishment of Hybridoma Lines
  • Support Protocol 3: Cloning by Limiting Dilution
  • Support Protocol 4: Preparation of Cloning/Expansion Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunization to Produce Monoclonal Antibodies

  Materials
  • Antigen
  • Complete Freund's adjuvant (CFA; Sigma)
  • Animal: pathogen‐free mouse, hamster, or rat (Armenian hamsters from Cytogen Research are recommended; see for discussion of animal choice)
  • Incomplete Freund's adjuvant (IFA; Sigma), optional
  • 1‐ to 2‐ml glass syringes with Luer‐Lok tips, sterile
  • 3‐way stopcock
  • 20‐ and 22‐G needles, sterile
  • Additional reagents and equipment for intraperitoneal injection (Donovan and Brown, )CAUTION: CFA is an extremely potent inflammatory agent, particularly if introduced intradermally or into the eyes. Profound sloughing of skin or loss of sight may occur. Self‐injection can cause a positive TB skin test and lead to a granulomatous reaction. Use gloves and protective eyewear when handling CFA.

Basic Protocol 2: Cell Fusion and Selection of Hybridomas

  Materials
  • SP2/0‐Ag14 myeloma cell line (drug‐marked, nonsecretory; ATCC #CRL 1581)
  • Complete DMEM‐10 and ‐20 media ( appendix 2A) with 10 mM HEPES and 1 mM sodium pyruvate
  • Primed animal; mouse, hamster, or rat (10 to 14 days after primary immunization; see protocol 1)
  • Complete DMEM medium ( appendix 2A), serum‐free
  • 50% polyethylene glycol (PEG), sterile
  • Ammonium chloride solution (see recipe)
  • recipeComplete DMEM‐20 ( appendix 2A) with 10 mM HEPES, 1 mM sodium pyruvate, and 1× HAT or 1× HT supplement
  • 175‐cm2 flasks
  • Fine‐mesh metal screen
  • 50‐ml conical polypropylene centrifuge tubes
  • Beckman TH‐4 rotor or equivalent
  • 96‐well flat‐bottom microtiter plates
  • Additional reagents and equipment for counting cells and assessing cell viability by trypan blue exclusion ( appendix 3A)

Support Protocol 1: Screening Primary Hybridoma Supernatants

  Additional Materials
  • Growing hybridomas ( protocol 2)
  • Additional reagents and equipment for ELISA (Hornbeck, ) and indirect immunofluorescence (unit 6.12)

Support Protocol 2: Establishment of Hybridoma Lines

  Additional Materials
  • Growing hybridomas ( protocol 2)
  • Cloning/expansion medium ( protocol 6)
  • 24‐well microtiter plates
  • Additional reagents and equipment for cryopreservation of cells ( appendix 3B)

Support Protocol 3: Cloning by Limiting Dilution

  Additional Materials
  • Candidate hybridoma line ( protocol 4)
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Figures

Videos

Literature Cited

   Bazin, R. and Lemieux, R. 1989. Increased proportion of B cell hybridomas secreting monoclonal antibodies of desired specificity in cultures containing macrophage‐derived hybridoma growth factor (IL‐6). J. Immunol. Methods 116:245‐249.
   Bonifacino, J.S., Dell'Angelica, E.C., and Springer, T.A. 2001. Immunoprecipitation. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 8.3.1‐8.3.28 John Wiley & Sons, Hoboken, N.J.
   Coffino, P., Baumal, R., Laskov, R., and Scharff, M.D. 1972. Cloning of mouse myeloma cells and detection of rare variants. J. Cell. Physiol. 79:429‐440.
   Donovan, J. and Brown, P. 2006. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1‐1.6.9. John Wiley & Sons, Hoboken, N.J.
   Goding, J.W. 1986. Monoclonal Antibodies: Principles and Practice. Academic Press, San Diego.
   Harlow, E. and Lane, D. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Hornbeck, P. 1991. Enzyme‐linked immunosorbent assays. In Current Protocols in Immunology (J.E. Coligan, B.E. Bierer, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.1.1‐2.1.22. John Wiley & Sons, Hoboken, N.J.
   Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined pecificity. Nature (Lond.) 256:495‐497.
   Logdberg, L., Gunter, K.C., and Shevach, E.M. 1985. Rapid production of monoclonal antibodies to T lymphocyte functional antigens. J. Immunol. Methods 79:239‐249.
   Nordan, R.P. and Potter, M. 1986. A macrophage‐derived factor required by plasmacytomas for survival and proliferation in vitro. Science 233:566‐568.
   Raybould, T.J.G. and Takahashi, M. 1988. Production of stable rabbit‐mouse hybridomas that secrete rabbit MAb of defined specificity. Science 240:1788‐1790.
   Sanchez‐Madrid, F., Szklut, P., and Springer, T.A. 1983. Stable hamster‐mouse hybridomas producing IgG and IgM hamster monoclonal antibodies of defined specificity. J. Immunol. 130:309‐317.
   Schreiber, R.D., Hicks, R.D., Celada, A., Buchmeier, N.A., and Gray, P.W. 1985. Monoclonal antibodies to murine γ‐interferon which differentially moderate macrophage activation and antiviral activity. J. Immunol. 134:1609‐1618.
   Sharpe, R.J., Schweizer, R.T., Krisiunas, L., Mihalyo, M.A., and Poow, L.M. 1985. Efficient production of T cell‐specific monoclonal antibodies through initial tolerance induction to nonspecific antigens. Transplant. Proc. 17:2757‐2759.
   van Lier, R.A., Boot, J.H., Verhoeven, A.J., de Groot, E.R., Brouwer, M., and Aarden, L.A. 1987. Functional studies with anti‐CD3 heavy chain isotype switch‐variant monoclonal antibodies. Accessory cell‐independent induction of interleukin‐2 responsiveness in T cells by epsilon‐anti‐CD3. J. Immunol. 139:2873‐2879.
Key References
   Goding, . 1986. See above.
  An in‐depth discussion of MAb production.
   Köhler and Milstein, 1975. See above.
  The first description of MAbs, for which the authors were awarded the Nobel Prize.
   Oi, V.T. and Herzenberg, L.A. 1980. Immunoglobulin‐producing hybrid cell lines. In Selected Methods in Cellular Immunology (B.B. Mishell and S.M. Shiigi, eds.) pp. 351‐372. W.H. Freeman, New York.
  This reference is the basis for this protocol and for the development of many MAbs in the literature.
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