Selective photocrosslinking of functional ligands to antibodies viatheconserved nucleotide binding site

Nathan J. Alves, Matthew M. Champion, Jared F. Stefanick, Michael W. Handlogten, Demetri T. Moustakas, Yunhua Shi, Bryan F. Shaw, Rudolph M. Navari, Tanyel Kiziltepe, Basar Bilgicer

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

The conserved nucleotide binding site (NBS), found in the Fab variable domain of all antibody isotypes, remains a not-so-widely known and under-utilized site. Here, we describe a UV photocrosslinking method (UV-NBS) that utilizes the NBS for site-specific covalent functionalization of antibodies, while preserving antibody activity. We identified a small molecule, indole-3-butyric acid (IBA), which has affinity for the NBS (Kd=1-8μm) and can be photocrosslinked to antibodies upon UV energy exposure. By synthesizing their IBA conjugated versions, we have successfully photocrosslinked various types of functional ligands to antibodies at the NBS, including affinity tags (biotin), fluorescent molecules (FITC), peptides (iRGD), and chemotherapeutics (paclitaxel). An optimal UV exposure of 1-2J/cm2 yielded the most efficient photocrosslinking and resulted in 1-2 conjugations per antibody, while preserving the antigen binding activity and Fc related functions. Analysis of the photocrosslinked conjugates using western blotting, mass spectrometry, and computational docking simulations demonstrated that the photocrosslinking specifically takes place at the Y/F42 residue in framework region 2 of the antibody light chain. Taken together, the UV-NBS method provides a practical, site-specific, and chemically efficient method to functionalize antibodies with significant implications in diagnostic and therapeutic settings.

Original languageEnglish (US)
Pages (from-to)5700-5710
Number of pages11
JournalBiomaterials
Volume34
Issue number22
DOIs
StatePublished - Jul 1 2013
Externally publishedYes

Keywords

  • Crosslinking
  • ELISA
  • Flow cytometry
  • Immunochemistry
  • Immunoglobulin
  • Molecular modeling

ASJC Scopus subject areas

  • Bioengineering
  • Ceramics and Composites
  • Biophysics
  • Biomaterials
  • Mechanics of Materials

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  • Cite this

    Alves, N. J., Champion, M. M., Stefanick, J. F., Handlogten, M. W., Moustakas, D. T., Shi, Y., Shaw, B. F., Navari, R. M., Kiziltepe, T., & Bilgicer, B. (2013). Selective photocrosslinking of functional ligands to antibodies viatheconserved nucleotide binding site. Biomaterials, 34(22), 5700-5710. https://doi.org/10.1016/j.biomaterials.2013.03.082