Competitive Binding Assay.

Polyacrylamide-based competitive binding assay (31) was used to evaluate the binding affinity of Siglec-8 for 6′S sLe^x. Microtiter plates (F96 MaxiSorp, Nunc) were coated with 100 µL per well of a 10 µg/mL solution of Siglec-8 lectin domain in assay buffer (20 mM Hepes, pH 7.4; 150 mM NaCl; and 1 mM CaCl₂), overnight at 4 °C. The coating solution was discarded, and the wells were blocked with 150 µL per well of 3% BSA in assay buffer for 2 h at 4 °C. After three washing steps with assay buffer (150 µL per well), a threefold serial dilution of the 6′S sLe^x ligand (50 µL per well) in assay buffer and streptavidin-peroxidase coupled polyacrylamide glycopolymer [6′S sLe^x-PAA (Lectinity), 50 µL per well of a 0.3 µg/mL solution] were added. The plate was incubated on a thermoshaker (PHMP-4, Grant Instruments) for 3 h at 25 °C and shaking speed 350 rpm and then carefully washed four times with 150 µL per well assay buffer. After the addition of 100 µL per well of the horseradish peroxidase substrate 2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonic acid), the colorimetric reaction was allowed to develop for 3 min, then stopped by the addition of 2% aqueous oxalic acid, before the optical density was measured at 415 nm on a microplate-reader (Spectramax 190, Molecular Devices). The IC₅₀ value of 6′S sLe^x was calculated with the Prism software (GraphPad Software, Inc.). The IC₅₀ defines the molar concentration of the test compound that reduces the maximal specific binding of 6′S sLe^x-PAA polymer to Siglec-8 by 50%.