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Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies

Nature Protocols volume 8, pages 1125–1148 (2013)Cite this article

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Abstract

This protocol describes the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant antigen-binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.

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Figure 1: IgG and recombinant antibody fragments.
Figure 2
Figure 3: Collier de Perles examples of antibody variable domains.
Figure 4: Highlight of the FR1 and FR4 most encountered amino acids for VH and V-κ chains.
Figure 5
Figure 6: Step-by-step expected results.

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Acknowledgements

C.F., G.U.L. and J.M. were supported by the EU (MagPro2Life Consortium), Science Foundation Ireland (grant no. 08/IN/2972) and University College Dublin. D.O. was supported by Science Foundation Ireland (grant no. SFI12/IP/1260). P.B. was supported by grant no. 07-EMPB-002-01 from the Agence Nationale de la Recherche.

Author information

Authors and Affiliations

  1. UCD Centre for Nanomedicine, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland

    Conor Fields, Gil U Lee & Julien Muzard

  2. Conway Institute, University College Dublin, Dublin, Ireland

    David O'Connell

  3. Protein Department, Genscript, Piscataway, New Jersey, USA

    Sujing Xiao

  4. School of Pharmacy, University Paris-Sud and Muséum National d'Histoire Naturelle–Centre National de la Recherche Scientifique,

    Philippe Billiald

  5. UMR 7245, Paris, France

    Philippe Billiald

  6. Chaire de Bioinformatique, Conservatoire National des Arts et Métiers, Paris, France

    Julien Muzard

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  1. Conor Fields
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  4. Gil U Lee
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Contributions

This manuscript is based on the protocols developed in our research laboratories from 2002 to 2013. All authors contributed to writing the manuscript and approved its final version.

Corresponding author

Correspondence to Julien Muzard.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Annotated Fd chain sequences with native leader sequences, FR and CDR according to IMGT (continued line) and Kabat (dotted line) numbering schemes. (PDF 733 kb)

Supplementary Figure 2

Annotated light chain sequences with native leader sequences, FR and CDR according to IMGT (continued line) and Kabat (dotted line) numbering schemes. (PDF 719 kb)

Supplementary Figure 3

Example of a 3D Fv homology model generated using [Box 1, OR12-13]. (PDF 981 kb)

Supplementary Figure 4

Example of an epitope-paratope interaction prediction generated using [Box 1, OR12-13]. (PDF 458 kb)

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Fields, C., O'Connell, D., Xiao, S. et al. Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies. Nat Protoc 8, 1125–1148 (2013). https://doi.org/10.1038/nprot.2013.057

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