Enzyme-Linked Carbohydrate-Binding Assay
Binding properties of bovine SPM to carbohydrates were analyzed by an enzyme-linked carbohydrate-binding assay (ELCBA). Polystyrene 96-well microtiter plates were coated with SPM samples (protein range from 1 ng to 8 |xg per well) diluted in coating buffer (containing 0.1 M Na2CO3, pH 9.6) and incubated overnight at 4°C. All further procedures were carried out at room temperature. The coating buffer was discarded, and the wells were blocked for 10 min with 0.3% (v/v) Tween-20 in HBS, followed by 0.05% Tween-20, 1% BSA in HbS for 1 h.
After removing the blocking solution, 50 |xl of biotin-conjugated carbohydrates (fucose-PAA-biotin, LeA-PAA-biotin, mannan-BSA-biotin, or fucoidan-biotin) were allowed to bind to the coated plates for 2 h in incubation buffer (containing 0.05% [v/v] of Tween-20 in HBS) as added in serial dilution starting at 20 ng/well. After binding, the fluids were removed and the wells were washed five times in washing buffer (containing 10 mM Tris, 150 mM NaCl, 5 mM CaCl2, and 0.05% [v/v] Tween-20, pH 7.5). The amount of immobilized biotin conjugates was quantified using neutravidin-HRP.