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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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.
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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.
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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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DOI: https://doi.org/10.1038/nprot.2013.057
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