Encyclopedia of Experiments: Immunology
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Transcript
Begin with a functionalized glycochip featuring printed glycan sub-arrays organized into blocks of arrays.
Each spot in the sub-array represents a distinct glycan — a carbohydrate-based polymer with variations in monosaccharide composition, chain length, and branching pattern.
Treat the glycochip with a blocking buffer to block non-specific interaction sites.
Transfer the glycochip and introduce diluted mouse serum in a surfactant-supplemented buffer to minimize antibody aggregation. Incubate under agitation.
Agitation promotes uniform serum distribution, facilitating selective binding of anti-glycan antibodies to specific glycans based on distinct molecular characteristics.
Wash with decreasing concentrations of a surfactant-containing buffer to disturb non-specific binding and rinse.
Overlay the glycochip with anti-mouse biotin-conjugated secondary antibodies, interacting with pre-bound anti-glycan antibodies and wash.
Introduce fluorophore-labeled streptavidin, binding to biotinylated secondary antibodies. Wash and remove excess solution.
Scan the glycochip and observe for distinct fluorescence spots at various positions suggesting glycan-specific circulating antibodies in serum.
