Site-specific antibody labeling via endo-S2 mediated Fc glycan remodeling.

Site-specific labeling of antibodies is highly valuable for diagnostic and therapeutic applications. The chemoenzymatic Fc glycan remodeling method provides a platform for site-specific antibody functionalization without perturbing antigen recognition. In this chapter, we describe a one-pot enzymatic method to produce fluorophore- and biotin-labeled antibodies using S. pyogenes endoglycosidase S2 (Endo-S2) as the enzyme and functionalized disaccharide oxazolines as the substrate. This method enables the efficient installation of azide, biotin, or fluorophore functionalities at the Fc glycosylation site, yielding homogeneous antibody conjugates. Trastuzumab, a therapeutic monoclonal antibody, was chosen as the model antibody for this protocol but the platform should also be applicable for other recombinant IgG antibodies. This protocol offers a robust and modular approach for preparing site-specifically labeled antibodies for various applications.