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Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site.
Alves, Nathan J; Champion, Matthew M; Stefanick, Jared F; Handlogten, Michael W; Moustakas, Demetri T; Shi, Yunhua; Shaw, Bryan F; Navari, Rudolph M; Kiziltepe, Tanyel; Bilgicer, Basar.
Affiliation
  • Alves NJ; Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, IN 46556, USA.
Biomaterials ; 34(22): 5700-10, 2013 Jul.
Article in En | MEDLINE | ID: mdl-23601661
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 (K(d) = 1-8 µM) 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 1-2 J/cm(2) 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.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Ultraviolet Rays / Conserved Sequence / Cross-Linking Reagents / Antibodies / Nucleotides Type of study: Prognostic_studies Limits: Animals Language: En Journal: Biomaterials Year: 2013 Type: Article Affiliation country: United States

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Ultraviolet Rays / Conserved Sequence / Cross-Linking Reagents / Antibodies / Nucleotides Type of study: Prognostic_studies Limits: Animals Language: En Journal: Biomaterials Year: 2013 Type: Article Affiliation country: United States