3 sialyllactose

Girard's reagent T (GT), phenylhydrazine, 2,5-dihydroxybenzoic acid (DHB), lactose (Lac), ribonuclease B (RNase B), ovalbumin (chicken egg white albumin), bovine fetuin, 3’-sialyllactose (3’-SL, α2,3-linked sialic acid), 6’-sialyllactose (6’-SL, α2,6-linked sialic acid), 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) hydrochloride 1-hydroxybenzotriazole (HOBt) hydrate, DL-dithiothreitol (DTT), sodium dodecyl sulfate (SDS), β-cyclodextrin and peptide-N-glycosidase F (PNGase F; supplied glycerol free) were obtained from Sigma (St. Louis, MO, USA). HPLC-grade solvents (acetonitrile, ethanol) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Water was obtained through a Milli-Q water purification system (Millipore, Bedford, MA, USA). Nonporous graphitized carbon (150 mg/4 mL) solid phase extraction (SPE) columns were obtained from Alltech Associates (Deerfield, IL, USA). Sep-Pak C18 (3 cc) solid phase extraction (SPE) cartridges were obtained from Water Corporation (Milford, Massachusetts, USA). Human serum was obtained from a healthy volunteer in Xi'an Jiaotong Hospital. The research investigations were performed after obtained the informed consent and in accordance with an institutional review board protocol approved by the Human Research Ethic Committee at the Jiaotong Hospital. All other chemicals were purchased from Aladdin Inc. (Shanghai, China).

Virus receptor specificity was determined as previously described [16 (link)]. Three biotinylated sialylglycopolymers were used: 3′-sialyllactose (3′SL, Neu5Acα2-3Galβ1-4Glc), 6′-sialyllactose (6′SL, Neu5Acα2-6Galβ1-4Glc), and 6-sialyl-N-acetyllactosamine (6′-SLN, Neu5Acα2-6Galβ1-4GlcNAc) (Sigma). 96-well fetuin-coated (1 μg/well) plates were washed, blocked with PBS containing 1% bovine serum albumin (BSA), and incubated overnight with 32 HA units of influenza viruses at 4°C. Plates were washed, 100 μL of biotinylated glycans were added and incubated at 4°C for 2 h. The plates were washed and incubated with 100 μL of horse-radish peroxidase–conjugated streptavidin at 4°C for 1 h. After a final wash, 50 μL of the tetramethylbenzidine substrate was added and incubated for 10 min at room temperature. The reaction was stopped with 50 μL of 50 mM hydrochloric acid, and absorbance was measured at 450 nm.