Production of Monoclonal Antibody Supernatant and Ascites Fluid

Wayne M. Yokoyama1

1 Howard Hughes Medical Institute and Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.10
DOI:  10.1002/0471142727.mb1110s83
Online Posting Date:  July, 2008
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Abstract

This unit details methods for production of monoclonal antibodies. Two methods are given for production of hybridoma supernatants, including one for large‐scale production. A protocol for large‐scale production of hybridomas or cell lines is presented for use in isolation of cellular proteins. Finally, a method is given for producing and obtaining mouse ascites fluid containing monoclonal antibody. Recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council) on the use of animals for producing monoclonal antibodies are also discussed. These recommendations are designed to foster the judicious use of animals and to strongly encourage the use of in vitro methods whenever possible. Curr. Protoc. Mol. Biol. 83:11.10.1‐11.10.10. © 2008 by John Wiley & Sons, Inc.

Keywords: antibody production; monoclonal; ascites

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of a Monoclonal Antibody Supernatant
  • Alternate Protocol 1: Large‐Scale Production of Monoclonal Antibody Supernatant
  • Alternate Protocol 2: Large‐Scale Production of Hybridomas or Cell Lines
  • Basic Protocol 2: Production of Ascites Fluid Containing Monoclonal Antibody
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Production of a Monoclonal Antibody Supernatant

  Materials
  • Hybridoma of interest (see unit 11.7)
  • Complete DMEM‐10 medium ( appendix 3F)
  • 175‐cm2 tissue culture flasks
  • 50‐ml conical centrifuge tubes, sterile
  • Beckman TH‐4 rotor (or equivalent)

Alternate Protocol 1: Large‐Scale Production of Monoclonal Antibody Supernatant

  • Complete DMEM‐10 medium ( appendix 3F) with 5 to 10 mM HEPES, pH 7.2 to 7.4
  • 70% ethanol
  • 850‐cm2 roller flask
  • Roller apparatus in 37°C room or incubator
  • 250‐ml conical centrifuge tubes, sterile
  • Beckman JS‐5.2 rotor (or equivalent)

Alternate Protocol 2: Large‐Scale Production of Hybridomas or Cell Lines

  • Complete DMEM‐10 medium ( appendix 3F) with 5 to 10 mM HEPES, pH 7.2 to 7.4
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 22), 4°C
  • 850‐cm2 roller flask
  • Roller apparatus in 37°C room or incubator
  • 250‐ml conical centrifuge tubes, sterile
  • Beckman JS‐5.2 rotor (or equivalent)

Basic Protocol 2: Production of Ascites Fluid Containing Monoclonal Antibody

  Materials
  • Nude mice, 6 to 8 weeks old and specific‐pathogen free, or syngeneic host if mouse‐mouse hybridomas are injected
  • Pristane (2,6,10,14‐tetramethylpentadecane; Aldrich)
  • Hybridoma of interest (see unit 11.7)
  • Complete DMEM‐10 medium ( appendix 3F) with 10 mM HEPES and 1 mM sodium pyruvate
  • Phosphate buffered saline (PBS) or HBSS ( appendix 22), sterile and without FBS
  • 20‐ or 22‐G needle and 18‐G needle
  • 175‐cm2 tissue culture flask
  • 50‐ and 15‐ml polypropylene conical centrifuge tubes, sterile
  • Beckman TH‐4 rotor (or equivalent)
  • 56°C water bath
  • 0.45‐µm filter
  • Additional reagents and equipment for performing ELISA (unit 11.2) and counting cells (unit 11.5)
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Figures

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Literature Cited

Literature Cited
   Andrew, S.M. and Titus, J.A. 1997. Purification of immunoglobulin G. Curr. Protoc. Immunol. 21:2.7.1‐2.7.12.
   Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Donovan, J. and Brown, P. 2006b. Handling and restraint. Curr. Protoc. Immunol. 73:1.3.1‐1.3.6.
   Donovan, J. and Brown, P. 2006c. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Donovan, J. and Brown, P. 2007. Animal health assurance. Curr. Protoc. Immunol. 76:1.1.1‐1.1.3.
   Fitch, F.W., Gajewski, T.F., and Yokoyama, W.M. 1997. Diagnosis and treatment of mycoplasma‐contaminated cell cultures. Curr. Protoc. Immunol. 21:A.3E.1‐A.3E.4.
   Holmes, K.L., Lantz, L.M., Fowlkes, B.J., Schmid, I., and Giorgi, J.V. 2001a. Preparation of cells and reagents for flow cytometry. Curr. Protoc. Immunol. 44:5.3.1‐5.3.24.
   Holmes, K.L., Otten, G., and Yokoyama, W.M. 2001b. Flow cytometry analysis using the Becton Dickinson FACS Caliber. Curr. Protoc. Immunol. 49:5.4.1‐5.4.22.
   NRC. 1999. Monoclonal Antibody Production: A Report of the Committee on Methods of Producing Monoclonal Antibodies, Institute of Laboratory Animal Research, National Research Council. National Academy Press, Washington, D.C.
   Yokoyama, W.M. 1997. Cryopreservation of cells. Curr. Protoc. Immunol. 21:A.3G.1‐A.3G.3.
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