biotin conjugated maa ii b 1265  (Vector Laboratories)

Journal: The Journal of Clinical Investigation

Article Title: Pathobiont-driven antibody sialylation through IL-10 undermines vaccination

doi: 10.1172/JCI179563

Figure Lengend Snippet: ( A ) Effect of αIL-10 antibody treatment on IsdB antibody glycosylation. IsdB antibodies purified from mice in (treated with Ctrl IgG or αIL-10) and assessed by lectin ELISA to determine antibody glycan content. ( B and C ) Effect of IL-23– or IL-6–neutralizing antibody on IsdB antibody sialylation. Serum IsdB antibodies from IL-6 ( B ) or IL-23 ( C ) antibody-treated, Sa/IsdB vaccinated mice, per , were assessed for α-2,6 or α-2,3 sialylation by SNA and MAA lectin ELISA. ( D ) UPLC-FL analysis of N-glycans released from PNGaseF treatment of purified IsdB antibodies from mice in (treated with Ctrl IgG or αIL-10). ( E ) Pie charts showing percentage of N-glycans in Glycan schematics used here and in all other figures follow the recommended symbol nomenclature for glycans (SNFG). Glycan nomenclature: blue, N-acetylglucosamine (GlcNAc); yellow, galactose (Gal); green, mannose (Man); pale blue, sialic acid (Neu5Gc or Neu5Ac); red, fucose (Fuc); A, antennae; S, sialic acid; F, fucose; G, galactose. Bars represent group median; each point represents an individual mouse ( A – C ). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, 1-way ANOVA followed by Bonferroni’s multiple-comparison adjustment ( A – C ).

Article Snippet: The plates were then incubated with 100 μL of biotinylated SNA (0.25 μg/mL dilution, Vector Laboratories, B-1305-2), MAA (4 μg/mL dilution, Vector Laboratories, B-1265-1), ECA (1 μg/mL dilution, Vector Laboratories, B-1145-5), or PHA-L (1 μg/mL dilution, Vector Laboratories, B-1115-2) in TBS-T (Tris-buffered saline with 0.05% Tween-20) for 60 minutes at 37°C.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Pathobiont-driven antibody sialylation through IL-10 undermines vaccination

doi: 10.1172/JCI179563

Figure Lengend Snippet: ( A and B ) Effect of αIL-10 antibody treatment on anti-Sa immunity conferred by IsdA, FhuD2, and MntC vaccination in Sa-exposed mice, performed as in ( n = 7–10 per mouse group from 2 independent experiments). ( C – E ) Effect of αIL-10 antibody treatment on serum antibody sialylation after IsdA ( C ), FhuD2 ( D ), or MntC ( E ) vaccination in Sa-exposed mice, performed as in A and B , assessed by MAA and SNA lectin binding. Bars represent group median; each point represents an individual mouse; dashed lines indicate the limit of detection ( A and B ). Bar represents group median; each point represents an individual mouse ( C – E ). * P < 0.05; ** P < 0.01; **** P < 0.0001, 1-way ANOVA followed by Bonferroni’s multiple-comparison adjustment ( A – E ).

Article Snippet: The plates were then incubated with 100 μL of biotinylated SNA (0.25 μg/mL dilution, Vector Laboratories, B-1305-2), MAA (4 μg/mL dilution, Vector Laboratories, B-1265-1), ECA (1 μg/mL dilution, Vector Laboratories, B-1145-5), or PHA-L (1 μg/mL dilution, Vector Laboratories, B-1115-2) in TBS-T (Tris-buffered saline with 0.05% Tween-20) for 60 minutes at 37°C.

Techniques: Binding Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Pathobiont-driven antibody sialylation through IL-10 undermines vaccination

doi: 10.1172/JCI179563

Figure Lengend Snippet: ( A and B ) α-2,6 and α-2,3 sialylation of purified human serum Sa antibodies ( n = 9) as assessed by SNA and MAA lectin binding normalized to IgG titer. ( C ) Sialylation of purified human serum antibodies against M protein (M) or S protein (S) of Streptococcus pyogenes ( n = 18) as assessed by MAA and SNA lectin binding normalized to IgG titer. Gray dashed line indicates median SNA or MAA level of IsdB antibody. ( D ) Sialylation of purified human serum antibodies against P . aeruginosa CbpD or FliC ( n = 5) as assessed by MAA and SNA lectin binding normalized to IgG titer. H, healthy normal human; CF, subject with cystic fibrosis. Gray dashed line indicates median SNA or MAA level of IsdB antibody. ( E ) OPK of Sa (LAC) by primary mouse neutrophils in the presence of human IsdB antibodies treated with α2-3 neuraminidase or buffer control from human donors ( n = 9). ( F ) Correlation between purified human serum IsdB antibody ( n = 9) binding to MAA lectin and OPK of LAC. Green circle indicates IsdB antibodies treated with α-2,3 neuraminidase. Bars represent group median; each point represents an individual human donor; error bars represent means± SD ( A – D ). Each point represents an individual human donor ( E and F ). * P < 0.05; ** P < 0.01; *** P < 0.001, Student’s t test ( C – E ), 1-way ANOVA followed by Bonferroni’s multiple-comparison adjustment ( A and B ) or linear regression ( F ).

Article Snippet: The plates were then incubated with 100 μL of biotinylated SNA (0.25 μg/mL dilution, Vector Laboratories, B-1305-2), MAA (4 μg/mL dilution, Vector Laboratories, B-1265-1), ECA (1 μg/mL dilution, Vector Laboratories, B-1145-5), or PHA-L (1 μg/mL dilution, Vector Laboratories, B-1115-2) in TBS-T (Tris-buffered saline with 0.05% Tween-20) for 60 minutes at 37°C.

Techniques: Purification, Binding Assay, Control, Comparison