Glycanassure apts kit

Capillary electrophoresis was used to quantify the relative abundance of antibody glycan structures as previously described (44 (link)). Briefly, hyperimmune IgG was bound to protein G immobilized on magnetic beads (New England Biolabs) and then incubated with IdeZ protease (New England Biolabs) to release the IgG-Fab portion. The beads still bound with IgG-Fc were washed, and N-glycans were released from the IgG-Fc and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) using the GlycanAssure APTS Kit (Thermo Fisher Scientific), as described in the manufacturer’s protocol. Labeled glycans were loaded and analyzed by the 3500 Genetic Analyzer (Thermo Fisher Scientific). Peaks for sialylated, fucosylated, galactosylated, and bisected (GlcNAc) structures were identified. The relative amount of each glycan structure was determined by calculating the area under the curve (AUC), which was normalized to the loaded APTS dye, for each peak then divided by the total area of all peaks. The percent enrichment was determined using the formula: [-(relative abundance of CHIKV load IgG – relative abundance of CHIKV eluate IgG)/relative abundance in the CHIKV load IgG]*100.

Plasma samples were diluted 1:10 with PBS and incubated overnight at 4°C with biotinylated gp120 (clade AE) coupled to magnetic streptavidin beads (NEB, MA, USA). Unspecific antibodies were washed away with PBS, and IgG-Fc fragments were enzymatically generated using IdeZ (NEB). IgG-Fc glycans were released from the protein and APTS labeled with GlycanAssure APTS kit (Thermo Fisher) and analyzed on a 3500xL genetic analyzer (Thermo Fisher) capillary electrophoresis instrument. Glycans were assigned based on retention times of known standard glycans as described before (52 (link)).