Journal: bioRxiv

Article Title: Neonatal Fc receptor is a functional receptor for human astrovirus

doi: 10.1101/2022.11.13.516297

Figure Lengend Snippet: (A) Anti- FCGRT antibodies block HAstV1 infection. Caco2 cells were cultured with 2, 0.66, or 0.2 µg/mL of anti- FCGRT antibody (NBP1-89128: NOVUS) for 1 hour before infection. The Caco2 cells were incubated with the activated virus (MOI 0.005) and anti- FCGRT at the indicated concentrations for 1 hour. The cells were then washed with fresh medium three times and cultured for 3 days with anti- FCGRT at the indicated concentrations. Error bars are mean ± SEM (n = 3). Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test (*, P < 0.05; ns, not significant). (B) ELISA measuring the binding affinity of FcRn and HAstV1 or 4. Immobilized recombinant FcRn was incubated with serial dilutions of each HAstV. HAstV captured by FcRn was detected by an anti-astrovirus antibody (8E7). Error bars are mean ± SEM (n = 10). Data are representative of two independent experiments. Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test (****, P < 0.0001; **, P < 0.01; ns, not significant). (C) Direct antigen ELISA measuring the binding affinity of anti-HAstV to immobilized purified HAstV. Each immobilized HAstV was detected by an anti-astrovirus antibody (8E7). Error bars are mean ± SEM (n = 5). (D) Each type of His-tagged VP27 was expressed by BL21. Purified His-tagged VP27 (2.5 µg) was immobilized and incubated with diluted recombinant FcRn. FcRn bound to VP27 was detected by an anti-FcRn antibody. Data are representative of two independent experiments. Error bars are mean ± SEM (n = 3). Significance was determined by one-way ANOVA with Dunnett’s multiple comparison test (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). Each type of immobilized His-tagged VP27 was detected with HRP-conjugated anti-His antibody (MBL), as shown in the right panel.

Article Snippet: ELISA POD Substrate TMB Solution (Nacalai Tesque, 05299-54) was used as a substrate, and the absorbance at 450 nm was detected using Ensight (PerkinElmer).

Techniques: Blocking Assay, Infection, Cell Culture, Incubation, Virus, Comparison, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Purification