Production of Monoclonal Antibodies

Wayne M. Yokoyama1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 16.1
DOI:  10.1002/0471143030.cb1601s03
Online Posting Date:  May, 2001
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Abstract

Monoclonal antibodies are particularly specific for a single epitope and can be produced in large quantities. When immune splenic cells are fused with a tumor cell line, they produce hybridomas each secreting one highly specific antibody. These cells are then characterized and cloned to allow production over time. This unit describes the production of monoclonal antibodies beginning with basic protocols for 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
  • 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, rat, or hamster (Armenian hamsters from Cytogen Research are recommended); see Critical Parameters 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
CAUTION: CFA is an extremely potent inflammatory agent, and is hazardous to the investigator, 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)
  • recipeComplete DMEM‐10 and ‐20 media 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 (see recipe)
  • Ammonium chloride solution (see recipe)
  • Complete DMEM‐20 with 10 mM HEPES, 1 mM sodium pyruvate, and 1× HAT or 1× HT supplement (see recipe)
  • 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 (unit 1.1)
NOTE: All culture incubations should be performed in a humidified 37°C, 5%, CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Support Protocol 1: Screening Primary Hybridoma Supernatants

  • Growing hybridomas ( protocol 2)
  • Additional reagents and equipment for ELISA ( appendix 3A) and indirect immunofluorescence (unit 4.3)

Support Protocol 2: Establishment of Hybridoma Lines

  • Growing hybridomas ( protocol 2)
  • Cloning/expansion medium ( protocol 6)
  • 24‐well microtiter plates
  • Additional reagents and equipment for cryopreservation of cells (unit 1.1)

Support Protocol 3: Cloning by Limiting Dilution

  • Candidate hybridoma line ( protocol 4)
  • Cloning/expansion medium ( protocol 6)
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Figures

Videos

Literature Cited

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.
   Coffino, P., Baumal, . R., Laskov, R., and Scharff, M.D. 1972. Cloning of mouse myeloma cells and detection of rare variants. J. Cell. Physiol. 79:429‐1440.
   Donovan, J. and Brown, P. 1995. Animal health assurance. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.1.1‐1.1.3. John Wiley & Sons, New York.
   Goding, J.W. 1996. Monoclonal Antibodies: Principles and Practice 3rd ed., Academic Press, San Diego.
   Harlow, E. and Lane, D. 1999. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 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. 34:1609‐1618.
   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, 1996. See above.
  An in‐depth discussion of MAb production.
   Köhler and Milstein, 1975. See above.
  The first description of MAb, 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 MAb in the literature.
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