sars membrane protein antibody  (Bio-Techne corporation)

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Inactivated whole-virion (WV) vaccine retains the protein composition and the structure of the original virus. (A) Scheme of inactivated WV (Gamma strain) production. (B) VeroE6/TMPRSS2 cells were treated with medium as a negative control, inactivated WV (undiluted) and live virus (diluted 10 4 -fold with medium) at 37 °C for 3 days, followed by plaque counting. (C, D) Inactivated WV were validated using (C) SDS-PAGE; left: marker, right: sample and (D) western blotting; left: marker, right: S protein and M protein. Each experiment was performed in duplicate. (E) TEM micrograph of SARS-CoV-2 virus particles, Scale bar, 100 nm.

Article Snippet: After blocking in 5% skim milk (w/v) diluted in distilled water, the transferred membrane was washed thrice with PBST (0.05% Tween-20 in PBS) and incubated with mouse anti-SARS-CoV-2 (2019-nCoV) spike antibody (clone: #42; Sino Biological, Beijing, China) or rabbit anti-SARS membrane protein antibody (polyclonal; Novus Biologicals, Littleton, CO, USA).

Techniques: Virus, Negative Control, SDS Page, Marker, Western Blot

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–H) Mice were immunized twice with inactivated WV intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A–F) Endpoint titers of S protein-specific IgG in (A) serum, (B) bronchoalveolar lavage fluid (BALF), and (C) nasal wash, and endpoint titers of S protein-specific IgA in (D) serum, (E) BALF, and (F) nasal wash were evaluated. (G, H) Measurement of neutralization against vesicular stomatitis virus-based pseudotyped viruses displaying Gamma spike of SARS-CoV-2 in (G) serum samples and (H) nasal wash samples. The significance of differences in the (G) 50-fold-diluted serum samples and (H) 4-fold-diluted nasal wash samples was evaluated. The data is presented as mean ± SD. Each experiment was performed in duplicate. (A–H) n = 5 per group. (A–H) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: After blocking in 5% skim milk (w/v) diluted in distilled water, the transferred membrane was washed thrice with PBST (0.05% Tween-20 in PBS) and incubated with mouse anti-SARS-CoV-2 (2019-nCoV) spike antibody (clone: #42; Sino Biological, Beijing, China) or rabbit anti-SARS membrane protein antibody (polyclonal; Novus Biologicals, Littleton, CO, USA).

Techniques: Neutralization, Virus

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine protects against upper and lower respiratory tract infections caused by SARS-CoV-2. (A–E) Mice were immunized twice with inactivated whole-virion (WV) vaccine intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A) Mice were intranasally infected with SARS-CoV-2 MA10 (5 × 10 4 PFU) in 5 μL of PBS and the SARS-CoV-2 titer in nasal turbinate was assayed using the plaque assay. (B–E) Mice were intranasally infected with SARS-CoV-2 MA10 ( B, C ; 5 × 10 4 PFU/mouse, D, E ; 2 × 10 5 PFU/mouse) in 20 μL of PBS; (B, D) body weight loss and (C, E) survival were monitored. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (A) n = 5 per group. (B, C) n = 4–5 per group. (D, E) n = 10 per group. (A, B, D) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. (C, E) ** P < 0.01 vs. the control group as indicated by comparing Kaplan–Meier curves using the Log rank test.

Article Snippet: After blocking in 5% skim milk (w/v) diluted in distilled water, the transferred membrane was washed thrice with PBST (0.05% Tween-20 in PBS) and incubated with mouse anti-SARS-CoV-2 (2019-nCoV) spike antibody (clone: #42; Sino Biological, Beijing, China) or rabbit anti-SARS membrane protein antibody (polyclonal; Novus Biologicals, Littleton, CO, USA).

Techniques: Infection, Plaque Assay, Control

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Systemic priming by mRNA vaccine followed by intranasal boosting with inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–C) Mice were immunized intranasally (i.n.) with inactivated WV or intramuscularly (i.m.) with 1 μg of mRNA vaccine encoding SARS-CoV-2 Spike. (A) Graphical illustration of the experimental procedure. (B, C) Endpoint titers of S protein-specific (B) IgA in the nasal wash and (C) IgG in the serum were evaluated. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (B, C) n = 7–11 per group. (B, C) ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: After blocking in 5% skim milk (w/v) diluted in distilled water, the transferred membrane was washed thrice with PBST (0.05% Tween-20 in PBS) and incubated with mouse anti-SARS-CoV-2 (2019-nCoV) spike antibody (clone: #42; Sino Biological, Beijing, China) or rabbit anti-SARS membrane protein antibody (polyclonal; Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: iScience

Article Title: Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

doi: 10.1016/j.isci.2022.105316

Figure Lengend Snippet: Figure 1. SARS-CoV-2 S can mediate cell-cell fusion in a metalloproteinase-dependent manner (A) Schematics of the S glycoprotein and amino acid sequences at the S1/S2 and S20 cleavage sites of mutants used in this study. In green: fusion peptide, in orange and yellow: N- and O- heptad repeats respectively, in purple: transmembrane domain, in red: amino acids surrounding the cleavage sites. Arrow heads depict the cleavage site.

Article Snippet: Polyclonal SARS Membrane protein antibody was acquired from Novus Biologicals.

Techniques:

Journal: iScience

Article Title: Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

doi: 10.1016/j.isci.2022.105316

Figure Lengend Snippet: Figure 2. SARS-CoV-2 S can mediate viral entry using a metalloproteinase-dependent entry route in cells expressing high levels of MMP-2 and MMP-9 (A, B, and E) 293T-ACE2, Calu-3, and HT1080 transfected with ACE2, were pre-treated for 1 h with 25 mM Camostat, 10 mM E64d, 40 mM TAPI-2, 10 mM GIX or Vehicle (DMSO) followed by the addition of lentiviral pseudoviruses encoding LacZ and bearing the SARS-CoV-2 D614G S, SARS-CoV-1 S, or VSV-G. After 48 h, cells were fixed and stained with X-gal overnight at 37C and foci representing infected cells were counted. Relative infection was calculated as the number of foci in the indicated inhibitor treatment relative to vehicle treatment. Each bar graph shows the mean of triplicate values of 3 independent experiments with error bars showing SD. The impact of inhibitors on infection compared to vehicle was analyzed using a two-way ANOVA and Dunnett’s post-hoc analysis. P-value lower than 0.05 was used to indicate a statistically significant difference (****, p < 0.0001, ***, p < 0.001, **, p < 0.01, *, p < 0.05). (C) Relative mRNA levels of MMP-2, MMP-9, ADAM10, and ADAM17 in various cell lines were measured by RT-qPCR. The level of actin mRNA expression in each sample was used to standardize the data, and normalization on 293T gene expression was performed. (293T, 293T-ACE2, Calu-3, HT1080: n R 3). (D) Gelatin zymogram of 40 mg of protein from conditioned media (24 h) from indicated cell lines reveals secreted MMP-2 (72 kDa) and MMP-9 (92 kDa) activity, arrows indicate the pro- and active- MMP-2 or MMP-9. Representative image of 3 independent experiments. See also Figure S2.

Article Snippet: Polyclonal SARS Membrane protein antibody was acquired from Novus Biologicals.

Techniques: Expressing, Transfection, Staining, Infection, Quantitative RT-PCR, Gene Expression, Activity Assay

Journal: iScience

Article Title: Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

doi: 10.1016/j.isci.2022.105316

Figure Lengend Snippet: Figure 8. Proposed model of the different SARS-CoV-2 entry pathways SARS-CoV-2 entry is mediated by the activation of S via proteolytic cleavage by host proteases. In the cathepsin- dependent entry pathway, SARS-CoV-2 is internalized following ACE2 binding and trafficked to endosomes where cathepsin L can cleave and activate unprocessed S (S0) and processed S (S1/S2) for the activation of membrane fusion. In the TMPRSS2-dependent entry pathway, S0 and S1/S2 are activated by TMPRSS2 (or other surface serine proteases such as TMPRSS13) following ACE2 engagement, leading to membrane fusion at the cell surface. In the MMP-dependent entry pathway, only processed S (S1/S2) is activated via MMPs following ACE2 binding allowing membrane fusion.

Article Snippet: Polyclonal SARS Membrane protein antibody was acquired from Novus Biologicals.

Techniques: Activation Assay, Binding Assay, Membrane