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Efficient method for constructing comprehensive murine Fab antibody libraries displayed on phage

H Orum1, P S Andersen, A Oster

  • 1Royal Danish School of Pharmacy, Department of Biology, Copenhagen.

Nucleic Acids Research
|September 25, 1993
PubMed
Summary
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We developed efficient methods for creating and expressing phage display libraries of murine Fab antibody fragments. This system enables the isolation of antigen-binding clones from diverse sources.

Area of Science:

  • Molecular Biology
  • Immunology
  • Biotechnology

Background:

  • Phage display technology is crucial for antibody discovery.
  • Efficient construction of antibody fragment libraries is essential for robust screening.
  • Existing methods for gene amplification and assembly can be optimized.

Purpose of the Study:

  • To develop optimized methodologies for constructing and expressing comprehensive phage display libraries of murine Fab antibody fragments.
  • To improve key steps in gene amplification, assembly, and expression for antibody fragment libraries.
  • To demonstrate the utility of the developed system in isolating antigen-binding clones.

Main Methods:

  • Designed low-degeneracy PCR primers for amplifying heavy and light-chain immunoglobulin genes.

Related Experiment Videos

  • Developed a 'jumping PCR' technique for assembling gene fragments into a bi-cistronic operon.
  • Constructed expression vectors for fusing Fab gene cassettes with phage gIIIp protein.

    • Main Results:

    • Achieved efficient amplification of Fab gene fragments from diverse hybridoma cell lines.
    • Successfully assembled and recombined heavy and light-chain gene fragments using jumping PCR.
    • Demonstrated the system's efficacy by isolating antigen-binding clones from hybridomas and immunized mouse libraries.

    Conclusions:

    • The developed methodologies provide an efficient platform for constructing and expressing comprehensive phage display libraries.
    • The optimized PCR and assembly techniques facilitate the creation of functional antibody fragment libraries.
    • This system is effective for isolating specific antigen-binding antibody fragments for research and therapeutic applications.