Biolayer Interferometry (BLI) assay

The BLI assay was performed on an Octet Red96 (ForteBio) instrument. Recombinant Siglec-7-Fc and Siglec-9-Fc were loaded onto Octet AHC2 biosensors (Satorius, part #18–5142) at concentrations of 10 μg/mL and 5 μg/mL, respectively, in kinetics buffer (0.05% Tween-20 in PBS) for 2 min. Association of (8^bio)₄-SA and (9^bio)₄-SA tetramers was conducted by immersing the biosensors in the kinetic buffer at concentrations ranging from 25 to 1000 nM for 3 min. Association of anti-Siglec-7 (BioLegend, clone S7.7) and anti-Siglec-9 (BioLegend, clone K8) was conducted by immersing the biosensors in the kinetic buffer at concentrations ranging from 5 to 200 nM for 3 min. Dissociation was recorded in the kinetics buffer for 5 min. The recorded signals were corrected by subtracting the reference background and the K_D, k_on and k_off values were calculated using a global fit model with the Octet Data Analysis software.