Figure 1. Generation and characterization of 7-6IgA. (A) Experimental scheme for the generation of hybridoma cells. Briefly, OTII-Tg CD4+ T cells were transferred into CD3ε−/− mice given 1 mg/ml OVA in drinking water. Hybridoma cells were prepared with mucosal lymphocytes 6 wk after transfer. LPL, lamina propria lymphocytes; PP, Peyer’s patches. (B) ELISA measurement of total and anti-OVA IgA in fecal samples collected from CD3ε−/− mice transferred with OTII-Tg CD4+ T cells, as in A, and control C57BL6 WT type mice, which received oral OVA. n = 3–19 mice for each point. (C) Competitive antigen-binding assay to measure OVA binding capacity. Purified 7-6IgA was preincubated with varying concentrations of OVA (OVA/IgA ratio in wt/vol is plotted on x axis), and the remaining OVA-free 7-6IgA was measured by ELISA. Arrow indicates the concentration of OVA sufficient to mask 7-6IgA antigen binding. Each dot represents one assay well, and the representative data of two similar experiments are shown. (D) Glycosylation of monoclonal 7-6IgA and 76-3IgG generated in A and 225.4IgA (B. theta–specific IgA) measured by periodic acid-Schiff (PAS) staining, and immunoblots showing equal antibody loading. The 7-6IgA data are representative of two similar experiments. (E) Normalized fluorescent signals from lectin microarray analysis of purified 7-6IgA, 225.4IgA, and 76-3IgG. The lectins for which the ratio of signal values both for 7-6IgA/76-3IgG > 2 and 7-6IgA/225.4IgA > 2 are shown in red, indicating glycans that may mediate 7-6IgA–specific bacterial coating. Fuc, fucose; NeuAc, N-acetylneuraminic acid (sialic acid); GlcNAc: N-acetylglucosamine; Gal, galactose; GalNAc, N-acetylgalactosamine. The data are representative of seven different concentrations of the antibodies measured under four different gain conditions. (F) The indicated cultured bacterial strains were incubated with 250 μg/ml of 7-6IgA, and the proportion of 7-6IgA–coated bacteria was measured by flow cytometry. n = 3–6 independent experiments for each group. (G) Flow cytometry measurements of B. theta coating with 7-6IgA, with and without preabsorption to OVA. Pooled data from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s multiple comparison test (F) or with Student’s t test (G; 7-6IgA versus 76-3IgG at the highest concentration). ***P < 0.001. Error bars represent SEM.
Figure 1.

Generation and characterization of 7-6IgA. (A) Experimental scheme for the generation of hybridoma cells. Briefly, OTII-Tg CD4+ T cells were transferred into CD3ε−/− mice given 1 mg/ml OVA in drinking water. Hybridoma cells were prepared with mucosal lymphocytes 6 wk after transfer. LPL, lamina propria lymphocytes; PP, Peyer’s patches. (B) ELISA measurement of total and anti-OVA IgA in fecal samples collected from CD3ε−/− mice transferred with OTII-Tg CD4+ T cells, as in A, and control C57BL6 WT type mice, which received oral OVA. n = 3–19 mice for each point. (C) Competitive antigen-binding assay to measure OVA binding capacity. Purified 7-6IgA was preincubated with varying concentrations of OVA (OVA/IgA ratio in wt/vol is plotted on x axis), and the remaining OVA-free 7-6IgA was measured by ELISA. Arrow indicates the concentration of OVA sufficient to mask 7-6IgA antigen binding. Each dot represents one assay well, and the representative data of two similar experiments are shown. (D) Glycosylation of monoclonal 7-6IgA and 76-3IgG generated in A and 225.4IgA (B. theta–specific IgA) measured by periodic acid-Schiff (PAS) staining, and immunoblots showing equal antibody loading. The 7-6IgA data are representative of two similar experiments. (E) Normalized fluorescent signals from lectin microarray analysis of purified 7-6IgA, 225.4IgA, and 76-3IgG. The lectins for which the ratio of signal values both for 7-6IgA/76-3IgG > 2 and 7-6IgA/225.4IgA > 2 are shown in red, indicating glycans that may mediate 7-6IgA–specific bacterial coating. Fuc, fucose; NeuAc, N-acetylneuraminic acid (sialic acid); GlcNAc: N-acetylglucosamine; Gal, galactose; GalNAc, N-acetylgalactosamine. The data are representative of seven different concentrations of the antibodies measured under four different gain conditions. (F) The indicated cultured bacterial strains were incubated with 250 μg/ml of 7-6IgA, and the proportion of 7-6IgA–coated bacteria was measured by flow cytometry. n = 3–6 independent experiments for each group. (G) Flow cytometry measurements of B. theta coating with 7-6IgA, with and without preabsorption to OVA. Pooled data from three independent experiments. Statistical analyses were performed with one-way ANOVA with Tukey’s multiple comparison test (F) or with Student’s t test (G; 7-6IgA versus 76-3IgG at the highest concentration). ***P < 0.001. Error bars represent SEM.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal