The relative abundance of antibody glycan structures was quantified by capillary electrophoresis, as previously described(36 (link)). Briefly, antigen-specific antibodies were purified using gp120-coupled magnetic beads (New England BioLabs), then treated with IdeZ protease (New England BioLabs) to collect the antibody Fc portion. N-glycans were removed from the Fc domains and labeled with APTS using the GlycanAssure™ APTS Kit (Thermo Fisher) as described in the manufacturer’s protocol. Labeled glycans were loaded onto a 3500 Genetic Analyzer (Thermo Fisher). Peaks of 5 major glycan structures (G0, G1, G2, Bisecting and Sialylated) were identified, and the relative abundance of each structure was determined by calculating the area under the curve of each peak divided by the total area of all peaks.
Idez protease
Manufactured by New England Biolabs
IdeZ protease is an enzyme that specifically cleaves immunoglobulin G (IgG) antibodies between the Fc and Fab regions. It can be used to generate Fab and Fc fragments from IgG molecules for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Glycanassure apts kit, by Thermo Fisher Scientific (4 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Gp120 coupled magnetic beads, by New England Biolabs (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Captureselect lc lambda affinity matrix human, by Thermo Fisher Scientific (2 mentions)
Magnetic beads, by New England Biolabs (1 mentions)
Protein g magnetic beads, by New England Biolabs (1 mentions)
6 protocols using idez protease
1
Quantifying Antibody Fc Glycan Structures
2
Glycan Profiling of Antibody Fc Region
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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.
3
Generation and Purification of F(ab')2
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F(ab′)2 fragments were generated using the IdeZ protease (NEB), purified using CaptureSelect LC-lambda affinity matrix (human) (ThermoFisher), and concentrated with Amicon ultracentrifugal filter tubes (30K MWCO) according to manufacturers’ instructions. Digestion and purification were confirmed by SDS-PAGE using mini-Protean TGX precast gels (4% to 20%) (Bio-Rad).
4
Antibody Glycan Profiling by Capillary Electrophoresis
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The relative abundance of antibody glycan structures was quantified by capillary electrophoresis, as previously described (Mahan et al., 2015 (link)). Briefly, mAbs were purified using protein G magnetic beads (New England Biolabs) and then treated with IdeZ protease (New England Biolabs) to release the antibody Fab portion. The Fc-attached beads were washed, and N-glycans were removed from the Fc domains 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 onto the 3500 Genetic Analyzer (Thermo Fisher Scientific). Peaks for differentially galactosylated, fucosylated, bisected (GlcNAc), and sialylated structures were identified (13 peaks were reproducibly observed). The relative abundance of each glycan structure was determined by calculating the area under the curve, normalized for equal amounts of loaded APTS dye, of each peak divided by the total area of all peaks.
5
Quantification of Ab Glycan Structures
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The relative abundance of Ab glycan structures was quantified by capillary electrophoresis, as previously described (39 (link)). Briefly, antigen-specific Abs were purified using gp120-coupled magnetic beads (New England Biolabs) and then treated with IdeZ protease (New England Biolabs) to collect the Ab Fc portion. N-glycans were removed from the Fc domains and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) using the GlycanAssure APTS Kit (Thermo Fisher), as described in the manufacturer’s protocol. Labeled glycans were loaded onto the 3500 Genetic Analyzer (Thermo Fisher). Peaks of five major glycan structures (G0, G1, G2, bisecting, and sialylated) were identified. The relative abundance of each glycan structure was determined by calculating the area under the curve, normalized for equal amounts of loaded APTS dye, of each peak divided by the total area of all peaks.
6
Generation and Purification of F(ab')2
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+ Expand
F(ab′)2 fragments were generated using the IdeZ protease (NEB), purified using CaptureSelect LC-lambda affinity matrix (human) (ThermoFisher), and concentrated with Amicon ultracentrifugal filter tubes (30K MWCO) according to manufacturers’ instructions. Digestion and purification were confirmed by SDS-PAGE using mini-Protean TGX precast gels (4% to 20%) (Bio-Rad).
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