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Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site

Paper ID Volume ID Publish Year Pages File Format Full-Text
6369 485 2013 11 PDF Available
Title
Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site
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 = 1–8 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/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.

Keywords
Immunoglobulin; Crosslinking; Immunochemistry; ELISA; Molecular modeling; Flow cytometry
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Selective photocrosslinking of functional ligands to antibodies via the conserved nucleotide binding site
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 34, Issue 22, July 2013, Pages 5700–5710
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us