Figure 5.
Anti-OVA-323 antibodies specifically bind to OVA-Mac-EVs from OT-II mice. (A) OT-II mouse OVA-Mac-EVs were incubated on ELISA plate coated with anti-CD9 antibodies for 2 h at room temperature. Then, biotinylated anti-OVA-323 IgG or biotinylated isotype IgG were added to selected wells, and the plate was incubated overnight at 4 °C. Afterwards, streptavidin-HRP was added to each well, which was followed by 45 min incubation at room temperature and addition of TMB substrate. Reaction was stopped after 5 min with 1 M H3PO4, and absorbance was measured at 450 nm with a reference wavelength of 570 nm. Then, blanked absorbance values were normalized to mean blanked absorbance detected in wells with added OVA-Mac-EVs but not antibodies (n = 4). (B) OT-II mouse OVA-Mac-EVs were either incubated with biotinylated anti-OVA-323 IgG overnight at 4 °C, ultracentrifuged, and coated onto latex beads, or coated onto latex beads and incubated with biotinylated anti-OVA-323 IgG for an hour at room temperature. Then, both OVA-Mac-EV preparations were incubated with streptavidin-FITC, and analyzed with flow cytometry (n = 3). (C) OT-II mouse OVA-Mac-EVs were incubated with either biotinylated anti-OVA-323 IgG, or FITC-conjugated isotype IgG overnight at 4 °C. After ultracentrifugation, both OVA-Mac-EV preparations were coated onto latex beads, incubated with streptavidin-FITC and fluoresceinated antibodies against CD9 and I-A/I-E, and analyzed with flow cytometry (n = 3). Data are expressed as mean ± SD. One-way or two-way ANOVA with post hoc RIR Tukey test; * p < 0.05, ** p < 0.01, *** p < 0.005.