Abstract
We present here a modular click chemistry-mediated method for the site-specific enzymatic labeling of essentially any antibody on the heavy chain N-linked glycans. While standard antibody labeling techniques can be cumbersome and tedious, with variable reproducibility and low yields, the described approach results in highly reproducible labeling with built-in robustness. The site-selective approach prevents disruption of the antigen binding domain that often occurs with standard random amine or thiol labeling methods. This is especially important when labeling monoclonal antibodies with vulnerable amino acids in or around the antigen binding domain. Additionally, the modular “Lego-like” labeling system allows for the simple attachment of numerous detection molecules including fluorescent proteins, biotin, fluorescent dyes, and other small molecule compounds.
The enzymatic labeling approach involves the use of a mutant modified beta-Gal-T1 enzyme, Gal-T(Y289L), that is substrate permissive and specifically labels terminal N-acetylglucosamine (GlcNAc) residues on N-linked antibody sugars with azide-modified N-acetylgalactosamine (GalNAz). Once labeled, the azide-tagged antibodies are reacted with dibenzocyclooctyne (DIBO)-functionalized molecules in a copper-free click reaction.
We demonstrate here the labeling of azide-tagged antibodies with DIBO-modified fluorescent proteins, fluorescent dyes, and other small molecule compounds. Antibody conjugates are efficiently produced with high reproducibility as determined by HPLC and gel electrophoresis. Flow cytometry applications data demonstrate high reproducibility and superior flow cytometry staining indices. Finally, absolute site-specificity of the labeling method is demonstrated using endoglycosidase treatment of labeled antibodies.