Serum samples were heat inactivated at 56o C for one hour, then spun down at >20,000 g to remove debris. Samples were pre-cleared by incubation with streptavidin-coated magnetic beads (NEB) for 1 hour, rotating. Antigens (wild type H3 A/Victoria/361/2011 and N2 A/Texas/50/2012) were biotinylated for ADCP and coupled to streptavidin-coated magnetic beads. Antigen-coated beads and pre-cleared samples were incubated for one hour at 37 C, rotating. Samples were then washed three times and resuspended in IDEZ (NEB) to cleave at the Fc-Fab hinge. Fc glycans were isolated and labeled using the GlycanAssure kit (ThermoFisher) and APTS-labeled glycans were analyzed by capillary electrophoresis on a 3500xL genetic analyzer with GlycanAssure software (ThermoFisher Applied Biosystems, v2). Pre-labeled libraries are used as references. Results are reported for each identified peak as percentage of total isolated glycans, and samples without identifiable traces are excluded from the analysis. Peaks are combined into glycoform groups for visualization and statistical testing (e.g. combining all sialylated glycoforms).
Glycanassure kit
Manufactured by Thermo Fisher Scientific
The GlycanAssure kit is a laboratory product offered by Thermo Fisher Scientific. It is designed to facilitate the analysis of glycans, which are complex carbohydrate structures found in various biological molecules. The kit provides the necessary components and protocols for the preparation and detection of glycans.
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Lab products found in correlation
2 protocols using glycanassure kit
1
Fc Glycan Analysis of Antigen-Specific Antibodies
2
Glycan Analysis of Antigen-Specific Antibodies
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HA and NA antigens (H1 A/California/07/2009, H3 A/Texas/50/2012, N2 A/Texas/50/2012) were coated onto magnetic beads through biotin-streptavidin as for ADCP. Serum samples were pre-cleared with non-coupled magnetic beads to remove nonspecific serum binding components. Pre-cleared samples were incubated with antigen-coated beads for 2 hours at 37C to form immune complexes. Beads were washed and Fc regions were cleaved off from beads by IDEZ digestion (New England Biolabs). Resulting solubilized Fc regions were then deglycosylated and labeled with APTS per GlycanAssure kit instructions (Thermo Fisher). Labeled glycans were analyzed by capillary electrophoresis (GeneScan 3500XL, Applied Biosciences) and GlycanAssure software (ThermoFisher). Peaks were called based on known labeled glycan standards and reported as a percentage of total glycan peak area.
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