Journal: Bioconjugate chemistry

Article Title: Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies

doi: 10.1021/acs.bioconjchem.9b00273

Figure Lengend Snippet: Human IgG and IgA have pronounced recognition of Neu5Gc-glycans with minimal lot-to-lot variability. (A) Each IVIG brand and pooled IgA were examined at serial dilutions of 1, 0.5, 0.25, and 0.125 mg/mL total protein, at 100 μL/array, glycan binding reactivity was detected with Cy3-anti-human IgG (1.2 μg/mL) or Cy3-anti-human IgA (1.6 μg/mL), respectively, and then scanned, and relative fluorescent units (RFUs) were calculated. Binding patterns were examined in accordance with glycans type, skeleton, and linkage, as described in Table 1. (B) Mean RFU of the 4 dilutions of each IVIG brand and pooled IgA against all Neu5Gc-glycans and all Neu5Ac-glycans that are described in A (mean ± sem). (C) Glycan microarray analysis of seven IVIG-1 and two pooled IgA lots was tested at 0.5 mg/mL total protein, 100 μL/array (In IVIG-1, the Pearson correlation coefficient to lot1 was 0.93, 0.92, 0.85, 0.88, 0.9, 0.82, and 0.82 between the two lots of IgA.).

Article Snippet: Sialoglycan Microarray Fabrication Arrays were fabricated with NanoPrint LM-60 Microarray Printer (Arrayit) on epoxide-derivatized slides (Corning) with 16 subarray blocks on each slide.

Techniques: Binding Assay, Microarray

Journal: Bioconjugate chemistry

Article Title: Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies

doi: 10.1021/acs.bioconjchem.9b00273

Figure Lengend Snippet: Competitive glycan microarray inhibition assays of human anti-Neu5Gc IgG and IgA. IVIG (A) and pooled IgA (B) were analyzed. IVIG-1, -3, and -5 and pooled IgA were examined at 0.5 mg/mL total protein in PBS/OVA pH 7 or mixed with 2 mM of 2-O-methyl-α-Neu5Ac (Ac2Me), 2-O-methyl-α-Neu5Gc (Gc2Me) in PBS/OVA pH 7, at 100 μL/tube, and incubated on ice for 2 h. Then, samples were each examined on glycan microarrays, glycan binding reactivity was detected with Cy3-anti-human IgG (1.2 μg/mL) or Cy3-anti-human IgA (1.6 μg/mL), respectively, and then scanned, and relative fluorescent units (RFUs) were calculated. (A) RFU IgG reactivities against all Neu5Gc-glycans in the presence of Ac2Me, Gc2Me, or without inhibitor were calculated (Box and Whiskers showing Min to Max; one-way ANOVA, **** p < 0.0001). (B) RFU IgA reactivities against all Neu5Gc-/Neu5Ac-glycans in the presence of Ac2Me, Gc2Me, or without inhibitor were calculated (Box and Whiskers showing Min to Max; one-way ANOVA, **** p < 0.0001, *** p < 0.001, ** p < 0.01).

Article Snippet: Sialoglycan Microarray Fabrication Arrays were fabricated with NanoPrint LM-60 Microarray Printer (Arrayit) on epoxide-derivatized slides (Corning) with 16 subarray blocks on each slide.

Techniques: Microarray, Inhibition, Incubation, Binding Assay

Journal: Bioconjugate chemistry

Article Title: Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies

doi: 10.1021/acs.bioconjchem.9b00273

Figure Lengend Snippet: Differential inhibition of anti-Neu5Gc IgG and IgA reactivities. Glycan microarray binding of IVIG or serum IgA against all Neu5Gc-glycans was investigated with serial dilutions of 8 mM to 0.06 mM Ac2Me (A) or Gc2Me (B) or 0.9 mM to 0.025 mM Neu5Gc-glycopeptides, in PBS/OVA pH 7 (C). Inhibitors were serially diluted in PBS buffer (pH 7.0), and IVIG-1 or pooled IgA was added (0.5 mg/mL total protein, PBS/OVA; 100 μL/tube) and then incubated on ice for 2 h. Samples were then each examined on glycan microarrays, and binding was detected with Cy3-anti-human IgG (1.2 μg/mL) or Cy3-anti-human IgA (1.6 μg/mL) and then scanned, and relative fluorescent units (RFUs) were calculated. Statistical analysis was performed with Prism version 8, and outlier values were excluded. (A) Compared to binding with no inhibition, Ac2Me at 4 mM and 8 mM inhibits reactivity of anti-Neu5Gc IgA for each glycan, while IgG reactivity was not inhibited at all (**, p = 0.0011; ***, p = 0.0002; ns, respectively; two-way ANOVA, Dunnett post-test). (B) Compared to binding with no inhibition, Gc2Me inhibits both anti-Neu5Gc IgA/IgG reactivity (****, p < 0.0001; two-way ANOVA, Dunnett post-test). (C) Compared to binding with no inhibition, Neu5Gc-glycopeptides inhibit both anti-Neu5Gc IgA/IgG reactivity (****, p < 0.0001; two-way ANOVA, Dunnett post-test). (D) Normalized mean RFU comparing anti-Neu5Gc IVIG-1 and IgA shows that only in IgA Ac2Me inhibits reactivity against Neu5Gc-glycans, while IgG shows no inhibition (two-way ANOVA, Sidak post-test; 4 mM p = 0.0015, 8 mM p < 0.0001).

Article Snippet: Sialoglycan Microarray Fabrication Arrays were fabricated with NanoPrint LM-60 Microarray Printer (Arrayit) on epoxide-derivatized slides (Corning) with 16 subarray blocks on each slide.

Techniques: Inhibition, Microarray, Binding Assay, Incubation

Journal: Bioconjugate chemistry

Article Title: Differential Recognition of Diet-Derived Neu5Gc-Neoantigens on Glycan Microarrays by Carbohydrate-Specific Pooled Human IgG and IgA Antibodies

doi: 10.1021/acs.bioconjchem.9b00273

Figure Lengend Snippet: IVIG and pooled IgA contain diverse anticarbohydrate antibodies. Glycan microarray slides were treated with 50 mU/well AUS sialidase, washed, and then incubated with IVIG-1, IVIG-5, or pooled IgA (0.25 mg/mL at 100 μL/array). Glycan binding reactivity was detected with Cy3-anti-human IgG (1.2 μg/mL) or Cy3-anti-human IgA (1.6 μg/mL), respectively, and then scanned, and relative fluorescent units (RFUs) were calculated and analyzed (scatter plots show mean ± sem; paired t test, **** p < 0.00001).

Article Snippet: Sialoglycan Microarray Fabrication Arrays were fabricated with NanoPrint LM-60 Microarray Printer (Arrayit) on epoxide-derivatized slides (Corning) with 16 subarray blocks on each slide.

Techniques: Microarray, Incubation, Binding Assay