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Cloning of Variable Domains from Mouse Hybridoma by PCR

  • Nina Strebe
  • Frank Breitling
  • Dieter Moosmayer
  • Bodo Brocks
  • Stefan DübelEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Some hybridoma cell lines are very low producers, difficult to cultivate, or antibody production is lost upon prolonged culture. In these cases, a recombinant "hybridoma immortalization" can rescue a valuable antibody for further unlimited propagation in E. coli or other recombinant production systems. Further, monoclonal antibodies with promising specificity, but low affinity, can be improved by phage display, or mouse antibodies can be humanised for therapy. This chapter describes sets of thoroughly validated oligonucleotide primers for a PCR procedure to isolate and clone the DNA of antibody Fv fragments (antigen binding region) from mouse and rat hybridoma cell lines. The protocol includes a colony staining assay to identify E. coli clones producing scFv antibody fragments.

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Product Phage Display Library Hybridization Temperature scFv Fragment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Nina Strebe
    • 1
    • 2
  • Frank Breitling
    • 3
  • Dieter Moosmayer
    • 4
  • Bodo Brocks
    • 5
  • Stefan Dübel
    • 6
    Email author
  1. 1.Institut für Biochemie und BiotechnologieTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Sanofi-Aventis Deutschland GmbHFrankfurt am MainGermany
  3. 3.Karlsruhe Institute of Technology76344 Eggenstein-LeopoldshafenGermany
  4. 4.Bayer Schering Pharma AGBerlinGermany
  5. 5.Bodo Brocks, MorphoSys AGMartinsriedGermany
  6. 6.Institut für Biochemie und BiotechnologieTechnische Universität BraunschweigBraunschweigGermany

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