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This chapter describes the cloning strategy, expression, purification, and characterization of immunoglobulin G (IgG) and corresponding antibody fragments, namely fragment antigen-binding (Fab) and single-chain variable fragment (scFv). The examples use anti-HER2 antibody, Trastuzumab, as a standard antibody. The first method details a cloning strategy that enables the easy and quick reformatting of standard human IgG1 into antibody fragments using Gibson assembly. This approach generates two plasmids that bear either light chain or heavy chain genes, under the control of the CMV promoter. The second method, and as an alternative design, presents a single vector approach carrying both light and heavy chain genes, where both genes are separated by an internal ribosome entry site (IRES). Finally, this chapter outlines the transient transfection process in Chinese hamster ovary (CHO) mammalian cells, followed by antibody purification and characterization steps for laboratory applications, highlighting key notes to achieve optimal results for IgGs, Fabs, and scFvs.

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Transient Expression and Characterization of Antibodies and Antibody Fragments

  • 实验方案

  • 发表日期：

    2026年

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  • Greg del Val,
  • Florence Gauye,
  • Romain Ollier,
  • Damien Nevoltris

摘要

This chapter describes the cloning strategy, expression, purification, and characterization of immunoglobulin G (IgG) and corresponding antibody fragments, namely fragment antigen-binding (Fab) and single-chain variable fragment (scFv). The examples use anti-HER2 antibody, Trastuzumab, as a standard antibody. The first method details a cloning strategy that enables the easy and quick reformatting of standard human IgG1 into antibody fragments using Gibson assembly. This approach generates two plasmids that bear either light chain or heavy chain genes, under the control of the CMV promoter. The second method, and as an alternative design, presents a single vector approach carrying both light and heavy chain genes, where both genes are separated by an internal ribosome entry site (IRES). Finally, this chapter outlines the transient transfection process in Chinese hamster ovary (CHO) mammalian cells, followed by antibody purification and characterization steps for laboratory applications, highlighting key notes to achieve optimal results for IgGs, Fabs, and scFvs.