Production of Monoclonal Antibodies

Wayne M. Yokoyama1, Michelle Christensen2, Gary Dos Santos2, Diane Miller2, Jason Ho3, Tao Wu3, Michael Dziegelewski3, Francisca A. Neethling3

1 Washington University School of Medicine, St. Louis, Missouri, 2 StemCell Technologies, Inc., Vancouver, British Columbia, 3 Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.5
DOI:  10.1002/0471142735.im0205s102
Online Posting Date:  October, 2013
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Abstract

This unit describes the production of monoclonal antibodies beginning with immunization, 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. An alternate protocol describes cell fusion and one‐step selection and cloning of hybridomas utilizing a semi‐solid methylcellulose‐based medium (ClonaCell‐HY from StemCell Technologies). Curr. Protoc. Immunol. 102:2.5.1‐2.5.29. © 2013 by John Wiley & Sons, Inc.

Keywords: monoclonal antibodies; B cell hybridomas; limiting dilution; semi‐solid methylcellulose‐based medium

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunization Using Complete Freund's Adjuvant to Produce Monoclonal Antibodies
  • Alternate Protocol 1: Immunization Using Sigma Adjuvant System to Produce Monoclonal Antibodies
  • Basic Protocol 2: Cell Fusion and Selection of Hybridomas
  • Alternate Protocol 2: Cell Fusion, Selection, and Cloning of Hybridomas Using a Semisolid Medium (ClonaCell‐HY)
  • Alternate Protocol 3: Cell Fusion and Selection Using Electrofusion
  • Support Protocol 1: Screening Primary Hybridoma Supernatants
  • Support Protocol 2: Establishment of Hybridoma Lines
  • Support Protocol 3: Cloning by Limiting Dilution
  • Support Protocol 4: Recloning in Semisolid Medium (ClonaCell‐HY)
  • Support Protocol 5: Preparation of Cloning/Expansion Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunization Using Complete Freund's Adjuvant 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 Critical Parameters for discussion of animal choice and unit 1.1)
  • 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 handling and restraint of animals (unit 1.3) and injection of rodents (unit 1.6)
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.

Alternate Protocol 1: Immunization Using Sigma Adjuvant System to Produce Monoclonal Antibodies

  Additional Materials (also see protocol 1)
  • Antigen, at concentrations >1 mg/ml, ideally 2 mg/ml
  • Sigma Adjuvant System (Sigma S6322), reconstituted per manufacturer's instructions
  • Animal(s): Mice of preferred strain (e.g., Balb/C, C57Bl/6, CD1), three to five mice per cohort
  • 1‐ or 2‐ml syringes with 26‐G needles
  • Sterile microcentrifuge tube of adequate size to mix antigen and adjuvant 1:1
  • Additional reagents and equipment for ELISA (unit 2.1)

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)
  • Complete DMEM‐20/HEPES/pyruvate/HAT (or HT) medium (see recipe)
  • 175‐cm2 flasks
  • Fine‐mesh metal screen or 70 µm synthetic screen
  • 50‐ml conical polypropylene centrifuge tubes
  • Beckman TH‐4 rotor or equivalent
  • 96‐well flat‐bottom microtiter plates
  • Additional reagents and equipment for animal euthanasia (unit 1.8), spleen removal (unit 1.10), and counting cells and assessing cell viability by trypan blue exclusion ( appendix 3B)

Alternate Protocol 2: Cell Fusion, Selection, and Cloning of Hybridomas Using a Semisolid Medium (ClonaCell‐HY)

  Materials
  • Myeloma cell line (e.g., SP2/0, X63Ag8.653; available from ATCC)
  • ClonaCell‐HY Monoclonal Antibody Production Kit (StemCell Technologies, Inc.) containing:
    • Medium A—ClonaCell‐HY Pre‐Fusion Medium and Hybridoma Expansion Medium, 500 ml
    • Medium B—ClonaCell‐HY Fusion Medium, 500 ml
    • Medium C—ClonaCell‐HY Hybridoma Recovery Medium, 100 ml
    • Medium D—ClonaCell‐HY Hybridoma Selection Medium containing HAT, 90 ml
    • Medium E—ClonaCell‐HY Hybridoma Growth Medium containing HT, 500 ml
  • Polyethylene glycol—ClonaCell‐HY PEG Solution, pretested for cell fusion, 1.5 ml
  • Immunized mouse, 1 to 4 days after final antigen boost ( protocol 1)
  • 3% (v/v) acetic acid
  • Liquid nitrogen (optional)
  • Fetal bovine serum (FBS) containing 20% (v/v) DMSO
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • 100‐mm petri dishes
  • Fine‐mesh metal screen
  • Low‐speed tabletop centrifuge
  • 3‐ml and 12‐ml syringes
  • 25‐ and 75‐cm2 tissue culture flasks
  • 16‐G blunt‐ended hypodermic needles
  • 96‐ and 24‐well tissue culture plates
  • Cryotubes (e.g., Nunc)
  • Liquid nitrogen freezer (Dewar flask and canes to accommodate cryotubes; optional)
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B), animal euthanasia (unit 1.8), spleen removal (unit 1.10), preparing a single‐cell suspension of splenocytes (unit 3.1), counting cells using a hemacytometer ( appendix 3A), assaying for antigen production from hybridoma clones by ELISA (unit 2.1), flow cytometry (Chapter ), or immunoblotting (unit 8.10), and cryopreservation of cells ( appendix 3G)

Alternate Protocol 3: Cell Fusion and Selection Using Electrofusion

  Materials
  • Myeloma cell line (e.g., SP2/0, NS‐1, P3.X63Ag8.653, or FOX‐NY, available from ATCC)
  • Myeloma growth medium for the myeloma cell line chosen (see instructions provided by ATCC)
  • 4% (w/v) sodium hydroxide solution
  • Sterile reagent‐grade H 2O
  • Spor‐Klenz sterilizing solution (Steris Life Sciences, http://www.sterislifesciences.com/)
  • Serum‐free Dulbecco's modified Eagle medium (DMEM)
  • BTX Cytofusion medium (Harvard Apparatus)
  • Hybridoma selection medium such as DMEM‐20/HEPES/pyruvate/HAT ( appendix 2A)
  • 70% ethanol
  • 125‐cm2 tissue culture flasks
  • Hybrimune Hybridoma Production System with a fusion chamber appropriate for the scale of fusion
  • Computer
  • 50‐ml conical polypropylene centrifuge tubes96‐well tissue culture plates
  • Additional reagents and equipment for counting cells ( appendix 3A) and determining cell viability by trypan blue exclusion ( appendix 3B)

Support Protocol 1: Screening Primary Hybridoma Supernatants

  Additional Materials (also see protocol 3)
  • Growing hybridomas ( protocol 3)
  • Additional reagents and equipment for ELISA (unit 2.1) and indirect immunofluorescence (unit 5.3)

Support Protocol 2: Establishment of Hybridoma Lines

  Additional Materials (also see protocol 3)
  • Growing hybridomas ( protocol 3 or Alternate Protocols protocol 42 or protocol 53)
  • Cloning/expansion medium ( protocol 10)
  • 24‐well microtiter plates
  • Additional reagents and equipment for cryopreservation of cells ( appendix 3G)

Support Protocol 3: Cloning by Limiting Dilution

  Additional Materials (also see protocol 3)
  • Candidate hybridoma line ( protocol 7)

Support Protocol 4: Recloning in Semisolid Medium (ClonaCell‐HY)

  Additional Materials (also see protocol 4)
  • Growing hybridomas ( protocol 3 or Alternate Protocols protocol 42 or protocol 53)

Support Protocol 5: Preparation of Cloning/Expansion Medium

  Materials
  • Mice
  • Complete DMEM‐20/HEPES/pyruvate/HAT (or HT) medium (see recipe)
  • 75‐cm2 tissue culture dishes
  • 50‐ml conical polypropylene centrifuge tubes
  • 0.45‐µm filters
  • Additional reagents and equipment for thymectomy (unit 1.9) and preparation of single‐cell suspension from thymus (unit 3.1)
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Figures

  •   FigureFigure 2.5.1 Stages of monoclonal antibody production, with references to the basic, alternate, and support protocols in this unit (as well as subsequent units) that describe the steps.

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.
  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.
  Davis, J.M., Pennington, J.E., Kubler, A‐M., and Conscience, J.F. 1982. A simple, single‐step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods 50:161‐171.
  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.
  Kennett, R.H., McKearn, T.J., and Bechtol, K.B. 1980. Monoclonal Antibodies. Plenum, New York.
  Köhler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. 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.
  Zimmermann, U., Vienken, J., Halfmann, J., and Emeis, C.C. 1985. Electrofusion: a novel hybridization technique. Adv. Biotechnol. Processes 4:79‐150.
Key References
  Goding, 1986. 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. Immuno‐globulin‐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.
Internet Resources
  http://www.stemcell.com/en/Products/All‐Products/ClonaCellHY‐Hybridoma‐Kit.aspx
  Web site with further background and order information for ClonaCell‐HY.
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