Cloning of Hybridoma Cell Lines by Limiting Dilution

Steven A. Fuller1, Miyoko Takahashi1, John G.R. Hurrell1

1 Allelix Inc., Mississauga, Ontario
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.8
DOI:  10.1002/0471142727.mb1108s01
Online Posting Date:  May, 2001
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Abstract

Cloning by limiting dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90% of the wells containing single clones are positive for antibody production.

     
 
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Table of Contents

  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • HAT medium (unit 11.6)
  • HT medium (unit 11.7)
  • Complete culture medium (unit 11.5)
  • Polyvinyl or polystyrene 96‐well microtiter plates
  • 6‐ and 24‐well culture plates
  • 8% CO 2‐in‐air gas mixture
  • Humidified CO 2 incubator
  • Cryotubes ( Nunc #3‐63401)
  • 2‐, 5‐, and 10‐ml serological pipets
  • Multichannel pipet and tips
  • Inverted microscope
  • Additional reagents and equipment for preparing mouse feeder cells for fusion and cloning (unit 11.6), for ELISA screening (unit 11.4), and for estimating cell viability by trypan blue exclusion (unit 11.5)
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Figures

Videos

Literature Cited

Literature Cited
   Galfre, G. and Milstein, C. 1981. Preparation of monoclonal antibodies: Strategies and procedures. Meth. Enzymol. 73(B):3‐46.
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