α2 6 n sialyltransferase

Modified TRBCs were prepared as described (53 (link)) with slight modifications. Briefly, all sialic acids were removed from the surface of TRBC by incubation of 62.5 mL of 20% TRBC in PBS with 50 mU of VCNA (Roche, Almere, the Netherlands) in 8 mM calcium chloride at 37°C for 1 h. Complete removal of SAs was confirmed by loss of HA of treated TRBC using control viruses. Resialylation was done using 0.5 mU α2,3-(N)-sialyltraferase (Sigma-Aldrich, Zwijndrecht, the Netherlands) or 25 mU α2,6-(N)-sialyltransferase (Sigma-Aldrich) and 1.5 mM CMP-SA (Merck, Darmstadt, Germany) at 37°C in 75 mL for 2 h to produce α2,3-TRBC and α2,6-TRBC, respectively. After washing, the TRBCs were resuspended in PBS containing 1% BSA to a final concentration of 0.5%. Resialylation of either α2,3-SA or α2,6-SA was confirmed by HA using viruses with known receptor specificity. Viruses were tested in standard HA assay using native and resialylated TRBCs. In brief, twofold dilutions of virus were made in PBS. An equal volume of 0.5% TRBCs were added and incubated at 4°C for 1 h before reading the HA titer.

All sialic acids (SA) were removed from the surfaces of TRBCs by incubating 1% TRBCs in PBS with 50 mU of Vibrio cholerae NA (VCNA) (Roche) in 8 mM calcium chloride (CaCl₂) at 37°C for 1 h. After two washing steps with PBS, VCNA-treated TRBCs were resuspended in PBS containing 1% bovine serum albumin (BSA) to a final concentration of 0.5% TRBCs. Removal of SA was confirmed by observation of a complete loss of hemagglutination of the TRBCs by control influenza A viruses. Subsequently, resialylation was performed using 0.5 mU of α2,3-N-sialyltransferase (Sigma) or 25 mU of α2,6-N-sialyltransferase (Sigma) and 1.5 mM CMP-sialic acid (Merck) at 37°C for 2 h in a final volume of 75 μl to produce α2,3-TRBCs and α2,6-TRBCs, respectively. After two washing steps, the TRBCs were resuspended in PBS containing 1% BSA to a final concentration of 0.5% TRBCs. Resialylation was confirmed by hemagglutination of viruses with known receptor specificity: A/H5N1 IN/05 and A/H3N2 NL/13. The receptor specificity of A/H5N8 ck/NL/14 and A/H5N6 GZ/14 was tested by performing a standard hemagglutination assay with the modified TRBCs.

All sialic acid residues were enzymatically removed from cRBCs by incubation with the 50 mU Vibrio cholera neuraminidase (VCNA; Roche) in 8 mM calcium chloride at 37 °C for 1 h and then resialylated followed by using either α2,6-(N)-sialyltransferase or α2,3-(N)-sialyltransferase (Sigma) at 37 °C for 4 h³⁵. The receptor binding avidity of NC/02 or NC/02^HA149 virus were determined by performing standard HA assays using 0.5% modified cRBCs.