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Related Experiment Videos

A method for constructing reshaping single-domain antibody.

Ju-Long Cheng1, Xiang-Bin Wang, Zhong Zhang

  • 1Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

Yi Chuan Xue Bao = Acta Genetica Sinica
|August 17, 2002
PubMed
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This study presents a novel method for reshaping single-domain antibodies, simultaneously enhancing antigen binding affinity. The efficient technique successfully constructed high-affinity anti-CD28 single-domain antibodies using random mutagenesis and phage display.

Area of Science:

  • Biotechnology
  • Immunology
  • Protein Engineering

Background:

  • Developing novel single-domain antibodies (sdAbs) with enhanced antigen-binding affinity is crucial for therapeutic applications.
  • Existing methods for antibody reshaping often require complex sequence modeling and separate affinity optimization steps.

Purpose of the Study:

  • To demonstrate a novel, practical, and efficient method for constructing reshaped single-domain antibodies.
  • To achieve simultaneous reshaping and enhancement of antigen-binding affinity in a single procedure.

Main Methods:

  • Utilized homologous human antibody framework regions (FRs) and introduced mutations via overlap PCR with Taq DNA polymerase and high Mg2+ concentration.
  • Constructed a phage library from reshaped antibody genes and selected high-affinity binders through multiple rounds of panning.

Related Experiment Videos

  • Expressed selected antibodies in E. coli and measured antigen-binding affinity using ELISA.

    • Main Results:

    • Successfully constructed reshaped anti-CD28 single-domain antibodies with high antigen-binding affinity.
    • Selected antibodies retained high binding affinity after expression and refolding.
    • The developed method proved feasible and efficient for sdAb construction and affinity enhancement.

    Conclusions:

    • The novel method integrates antibody reshaping and affinity enhancement into a single, efficient process.
    • This approach offers a practical strategy for generating high-affinity single-domain antibodies for various applications.
    • The constructed anti-CD28 sdAbs demonstrate the efficacy of the method for therapeutic target engagement.