Engineering, Expression, Purification, and Application of Glycosaminoglycan-Specific Antibodies.

Cluster of differentiation markers have been transformative for defining cell populations and their functional states, but recent work indicates that finer granularity can be achieved by considering the diverse heparan sulfate structures presented on proteoglycans. Heparan sulfates (HSs) are long, unbranched polysaccharides of a repeating disaccharide composed of hexuronic acid and N-acetylglucosamine. HS is attached to core proteins via serine residues. Owing to multiple modifications of the sugar moieties by a set of specific Golgi-resident modification enzymes, HSs exhibit extraordinary variations in modification patterns. Because these patterns are non-template based, they display substantial heterogeneity, yet are expressed reproducibly with high spatiotemporal and tissue-specific selectivity. This complexity of HS modification is, in turn, believed to control extracellular receptor:ligand interactions in a cell-specific fashion. Conventional methods to study HS structure require specialized expertise and instrumentation, and remain challenging due to the considerable heterogeneity of even tissue-specific HSs. Here, we describe protocols for the production and implementation of a panel of anti-HS single-chain variable fragments (scFvs) based on scFvs originally reported in the 1990s and early 2000s. This panel is an attractive resource for detailed study of HS modification patterns in various physiological processes. Rather than determining HS structure by analytical means, we use this panel to define cells based on the scFvs they bind. We detail practical considerations, strategies, and protocols for the construction, development, expression, purification, and application of the anti-HS scFv panel from bacterial and mammalian expression systems. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of anti-HS scFvs from bacterial expression systems Basic protocol 2: Expression and purification of anti-HS scFvs from mammalian expression systems Basic Protocol 3: Sortase-mediated site-specific labeling of anti-HS scFvs Basic Protocol 4: In vitro HS detection by ELISA Basic Protocol 5: In vitro HS detection by flow cytometry using indirect labeling in live Jurkat E6-1 cells Alternate Protocol 1: In vitro HS detection by flow cytometry using indirect labeling in fixed Vero cells Alternate Protocol 2: In vitro HS detection by flow cytometry using direct labeling in live Vero cells Basic Protocol 6: In vitro HS detection by immunofluorescence in fixed adherent cells Alternate Protocol 3: In vitro HS detection by immunofluorescence in suspension cells prior to fixation.