Monoclonal Antibody Supernatant and Ascites Fluid Production

Wayne M. Yokoyama1

1 Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.6
DOI:  10.1002/0471142735.im0206s40
Online Posting Date:  May, 2001
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Abstract

Procedures detailing the production of monoclonal antibody supernatants, including the production of larger amounts (liters), are presented here. The protocol for large‐scale production of hybridomas or cells (e.g., for isolation of cellular proteins) involves a similar procedure. A method for producing and obtaining ascites fluid containing the monoclonal antibody is also presented. The unit ends with a set of recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council). The recommendations are designed to foster the judicious use of animals for the production of monoclonal antibodies and to strongly encourage the use of in vitro methods whenever possible.

     
 
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Table of Contents

  • 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 (unit 2.5)
  • Complete DMEM‐10 medium ( appendix 2A)
  • 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 2A) with 5 to 10 mM HEPES, pH 7.2 to 7.4
  • 70% ethanol
  • 850‐cm2 roller flask and roller apparatus
  • 250‐ml conical centrifuge tubes, sterile
  • Beckman JS‐5.2 rotor (or equivalent)

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

  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 (unit 2.5)
  • Complete DMEM‐10 medium ( appendix 2A) with 10 mM HEPES and 1 mM sodium pyruvate
  • PBSor HBSS ( appendix 2A), sterile and without FBS
  • 20‐ or 22‐G needle and 18‐G needle
  • 175‐cm2 tissue culture flask
  • Beckman TH‐4 rotor (or equivalent)
  • 50‐ and 15‐ml polypropylene conical centrifuge tubes, sterile
  • 56°C water bath
  • Additional reagents and equipment for handling animals (units 1.1 1.3), intraperitoneal injection (unit 1.6), euthanasia (unit 1.8), ELISA (unit 2.1), and counting cells and cryopreservation ( 3.NaN)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.
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Figures

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Literature Cited

Literature Cited
  National Research Council. 1999. Monoclonal Antibody Production. National Academy Press, Washington, D.C.
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