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Related Experiment Videos

An improved method for generating single-chain antibodies from hybridomas

P J Nicholls1, V G Johnson, M D Blanford

  • 1Biochemistry Section, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD 20892.

Journal of Immunological Methods
|September 27, 1993
PubMed
Summary

This study presents a novel PCR method to efficiently clone functional antibody variable region genes from hybridomas, overcoming issues with aberrant gene rearrangements. The technique enables rapid screening of single-chain antibodies for antigen binding before large-scale production.

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Methods in molecular medicine·2011

Area of Science:

  • Immunology
  • Molecular Biology
  • Biotechnology

Background:

  • Cloning antibody variable (VL) kappa genes from hybridomas can be challenging due to aberrant VJ recombination.
  • Aberrant transcripts can outnumber productive RNA, leading to non-functional protein products after PCR amplification.

Purpose of the Study:

  • To develop a reliable method for recovering and cloning functional antibody variable region genes from hybridoma cDNA.
  • To enable efficient screening of single-chain antibodies for antigen-binding activity prior to in vivo production.

Main Methods:

  • Utilized V gene family-specific primers to recover antibody variable region genes from hybridoma cDNA.
  • Spliced VL and VH genes using PCR into a 5'-VL-LINKER-VH-3' format for expression.
  • Employed an in vitro coupled transcription/translation system in rabbit reticulocyte lysate for protein expression and functional screening.

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Main Results:

  • Successfully cloned and expressed the single-chain form of OKT9 antibody, which targets the human transferrin receptor.
  • Demonstrated that aberrantly rearranged VL kappa genes, containing a premature stop codon, do not produce full-length protein in vitro.
  • Confirmed that the in vitro expressed single-chain antibody retains the antigen-binding properties of the parent antibody.

Conclusions:

  • The developed method effectively distinguishes functional from non-functional antibody variable region genes.
  • This approach provides a valuable tool for screening single-chain antibodies for desired functions in a high-throughput manner.
  • Facilitates the selection of correctly rearranged antibody genes for subsequent large-scale production.