The glycan binding assay was used to test the binding of GII.6 P particles to oligosaccharides using enzyme-linked immunosorbent assay (ELISA) as previously described (Cong et al., 2019 (link)). Briefly, 20 μg/well of P particles was coated onto microtiter plates at 4 °C overnight. After blocking with 5% nonfat milk, 0.2 μg/well of biotin-labeled polyacrylamide (PAA)-conjugated oligosaccharides were added, including A trisaccharides (A), B trisaccharides (B), H disaccharides (H), H type 1 (H1), H type 2 (H2), H type 3 (H3), Lewis a (Lea), Lewis x (Lex), Lewis b (Leb), Lewis y (Ley), type I precursor (Lec), and type II precursor (LacNAc) (GlycoTech, USA). After incubation, horseradish peroxidase (HRP)-conjugated streptavidin (1:1500 in PBS) (Abcam, USA) was added. The 3,3′,5,5′-tetramethylbenzidine (TMB) kit (BD Biosciences, USA) was used to detect the HRP activity. The reaction was stopped by the addition of 1 mol/L phosphoric acid, and the absorbance was measured at 450 nm (OD450).
3 3 5 5 tetramethylbenzidine tmb kit
Manufactured by BD
Sourced in United States
The 3,3′,5,5′-tetramethylbenzidine (TMB) kit is a laboratory reagent used for the detection and quantification of various analytes in a sample. It is commonly used in enzyme-linked immunosorbent assays (ELISA) and other colorimetric detection methods. The kit contains the necessary components to perform the TMB reaction, which produces a colored product that can be measured spectrophotometrically.
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2 protocols using 3 3 5 5 tetramethylbenzidine tmb kit
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Glycan Binding Assay of GII.6 P Particles
2
Oligosaccharide Binding Assay via ELISA
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Enzyme-linked immunosorbent assay (ELISA)-based oligosaccharide binding assays were conducted as previously described (Sun et al.2020 (link)). The GST-VP8* fusion proteins were coated onto microtiter plates at 20 μg per well with incubation at 4 °C overnight. Following blockage with 5% nonfat milk, synthetic oligosaccharides conjugated with polyacrylamide (PAA) and biotin were added at 0.2 μg per well. The following oligosaccharides were used in this study: Neu5Ac, Neu5Gc, Neu5Acα2-3Gal, Neu5Acα2-6Gal, siaLea, siaLex, siaLec, Neu5Acα2-3Galβ1-4GlcNAc (3’SLN), A/B disaccharides, H1, H2, lea, lex, leb, ley, mucin core 1–mucin core 6, and core 8 (GlycoTech, USA). Then, horseradish peroxidase (HRP)-conjugated streptavidin (Abcam) was added at 0.1 μg per well. For each step, the plates were incubated at 37 °C for 1 h, and washed five times with 0.5% PBS-Tween 20 buffer between steps. The color reactions were developed using a 3,3’,5,5’-tetramethylbenzidine (TMB) kit (BD Biosciences). The absorbance of 450 nm was measured.
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