Glycan Binding Assay for Norovirus P Proteins

Glycan binding assays were carried out as described previously [51 (link)] using oligosaccharides representing various HBGA types, well-defined human saliva samples from donors with type O, A, B, and non-secretor (N) blood types, and/or pig gastric mucin (PGM). Briefly, polyacrylamide-oligosaccharides (GlycoTech Inc) at 2 µg/mL, pretreated saliva samples (our lab stocks) at 1:1000 dilution, or type III PGM (Sigma-Aldrich) 5 µg/mL were coated on microtiter plates at 4°C overnight. After blocking with 5% (w/v) non-fat milk, the coated HBGAs were incubated with the wild type or mutant P proteins (10 µg/mL or indicated concentrations) for 60 min at 37°C. The bound P proteins were detected as described previously [21 (link),22 (link)], using an in-house hyperimmune guinea pig serum against huNoV VLPs [51 (link)].

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Yang Y., Xia M., Wang L., Arumugam S., Wang Y., Ou X., Wang C., Jiang X., Tan M., Chen Y, & Li X. (2019). Structural basis of host ligand specificity change of GII porcine noroviruses from their closely related GII human noroviruses. Emerging Microbes & Infections, 8(1), 1642-1657.

Publication 2019

type III PGM

Assays Dilution Donors Gastric mucin Glycan Guinea pig Human Milk Mutant proteins Oligosaccharides P proteins Polyacrylamide Saliva Serum

Corresponding Organization : University of Cincinnati Medical Center

Other organizations : University of Chinese Academy of Sciences, Nankai University

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Variable analysis

independent variables

Wild type or mutant P proteins (10 µg/mL or indicated concentrations)

dependent variables

Binding of P proteins to coated HBGAs

control variables

Polyacrylamide-oligosaccharides (GlycoTech Inc) at 2 µg/mL
Pretreated saliva samples (our lab stocks) at 1:1000 dilution
Type III PGM (Sigma-Aldrich) 5 µg/mL

positive controls

Well-defined human saliva samples from donors with type O, A, B, and non-secretor (N) blood types

negative controls

None explicitly mentioned

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