Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Codon-optimized full-length Spike protein gene (S F ), S1 subunit gene and the receptor-binding domain (RBD) plus envelope protein genes of SARS-CoV-2 with and without 21 amino acids honeybee melittin signal peptide [(msp) NH 2 -MKFLVNVALVFMVVYISYIYA-COOH] gene in the purple box, and 49 amino acids VSV G protein transmembrane domain and cytoplasmic tail [(Gtc) NH 2 -SSIASFFFIIGLIIGLFL VLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK-COOH] gene in the red box were inserted into the G and L gene junction of rVSV Ind and rVSV NJ . In addition, 25- nucleotides-long VSV intergenic junctions (5´-CATATGAAAAAAACTAACAGATATC-3´), in the green box, were inserted between genes to provide transcription termination, polyadenylation and the transcription reinitiation sequences. Recombinant viruses were rescued by VSV reverse genetics . pT7: Bacteriophage T7 promoter for DNA-dependent RNA polymerase. N : VSV Nucleocapsid Protein gene. P : VSV Phosphoprotein gene. M : VSV Matrix protein gene. G : VSV Glycoprotein gene. L : VSV Large protein, RNA-dependent RNA polymerase gene. l : Leader region in the 3´-end of the VSV genome. t : Trailer region in the 5´-end of the VSV genome. HDV: Hepatitis delta virus ribozyme encoding sequences. T7δ: Bacteriophage T7 transcriptional terminator sequences. nt: nucleotides. aa: amino acids.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Binding Assay, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To check the expression of SARS-CoV-2 RBD, S1, S F , and E proteins from the rVSV Ind -SARS-CoV-2 infected cells, BHK-21 cells were infected with the virus at an MOI of 6. After six hours incubation at 37°C, cell lysates were prepared and protein expression was determined by Western blot analysis. Cell lysates were loaded in 5 μg quantity for SDS-PAGE. RBD, S1, and S F proteins were detected by rabbit antibody against SARS-CoV-2 RBD. S2 protein was detected by rabbit antibody against SARS-CoV-2 S2. E protein was detected by rabbit antibody against SARS-CoV-2 E peptides. (A) Expression of RBD, S1, and S F with and without msp and Gtc. (B) Expression of S2 with and without Gtc. (C) Expression of E protein. (D) Expression of VSV Ind N, P, M, and G proteins. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Expressing, Infection, Incubation, Western Blot, SDS Page

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Incorporation of SARS-CoV-2 S F , S1, S2, and RBD with or without VSV Gtc into rVSV Ind particles was examined by infecting BHK-21 cells with rVSV Ind -SARS-CoV-2 at an MOI of 3. The rVSV Ind -SARS-CoV-2 infected cells were incubated at 31°C for 6 hrs. Infected cell lysates were prepared in lysis buffer (lanes 1, 2, and 5). Culture media from the infected cells was centrifuged at 500 x g for 10 minutes and supernatant was filtered through a 0.45 μm filter to remove cell debris. The filtered culture media was loaded onto 1 ml of 25% sucrose cushion and ultra-centrifuged at 150,900 x g for 3 hrs. Supernatant on top of the 25% sucrose cushion was collected to check the soluble proteins in the media (lanes 3 and 6). Pelleted samples were checked for proteins incorporated into VSV particles (lanes 4 and 7). We detected RBD, S1, and S F proteins by Western blot using an antibody against the SARS-CoV-2 RBD protein. S2 and S F proteins were detected by rabbit antibody against SARS-CoV-2 S2. (A) Detection of S F and S1 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (B) Detection of S F and S2 proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (C) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S F -Gtc or rVSV Ind -S F . (D) Detection of S1 protein in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-S1-Gtc or rVSV Ind -S1. (E) Detection of RBD proteins in cell lysate, concentrated culture media, and virus pellet from cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. (F) Detection of VSV Ind proteins in cell lysate, concentrated culture media, and virus pellet from the cells infected with rVSV Ind -msp-RBD-Gtc+E-Gtc or rVSV Ind -msp-RBD+E. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Infection, Incubation, Lysis, Western Blot

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: To examine immune responses in mice, it was first necessary to purify rVSV-SARS-CoV-2 viral particles by anion-exchange chromatography. One μg of the purified rVSV-SARS-CoV-2 was analyzed by SDS-PAGE and the presence of RBD, S1, S2, and S F was determined by Western blot analysis. (A) Detection of RBD, S1, and S F on VSV particles. (B) Detection of S2 and S F on VSV particles. (C) Detection of VSV Ind and VSV NJ proteins. (D) Depicted model of pseudotype recombinant VSV virions with three different forms of SARS-CoV-2 Spike proteins. rVSV pseudotypes are formed when rVSV-SARS-CoV-2 Spike proteins are expressed with the msp at the NH 2 -terminus and VSV Gtc at the COOH-terminus. Purple box: honeybee msp, red box: VSV Gtc.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Chromatography, Purification, SDS Page, Western Blot, Recombinant

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were prime immunized with rVSV Ind -SARS-CoV-2 and boost immunized with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Serum was collected to determine SARS-CoV-2 S1 protein-specific antibody levels by ELISA on day 13, one day before boost-immunization, and on day 27, two weeks after boost-immunization. (A) Prime-boost vaccination schedule. (B) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 7 PFU/mouse ( C) Spike(ΔTM)-specific IgG titer after the prime-boost vaccination with doses of 5X10 8 PFU/mouse. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost immunized with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. SARS-CoV-2 neutralization was determined by FRNT 50 assay. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005; ***, p< 0.001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Neutralization, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice were prime vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after the prime-vaccination. Serum was collected to determine the SARS-CoV-2 Spike(ΔTM) protein-specific antibody level by ELISA on day 13, one day before the boost-vaccination and on day 27, two weeks after the boost-vaccination. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p<0.005, ***, p<0.001, ****, p< 0.0001; ns, not significant). The data were presented as means with error bars of standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Mice were primed with rVSV Ind -SARS-CoV-2 followed with rVSV NJ -SARS-CoV-2 two weeks after prime-immunization. Two weeks after the boost-immunization, splenocytes were prepared and stimulated with a PepTivator SARS-CoV-2 Prot_S [ (A) ], or an irrelevant (control) peptide derived from the HIV Gag (B) . IFN-γ spot-forming units (SFUs) were enumerated by ELISPOT. Statistical significance was determined by two-way ANOVA with Tukey’s correction (*, p < 0.05; **, p < 0.005; ns, not significant). Data are presented as mean SFU numbers with error bars representing standard deviation (n = 5 mice per group). Purple box: honeybee msp, red box: VSV Gtc. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Derivative Assay, Enzyme-linked Immunospot, Standard Deviation, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Six-week-old female hACE2 transgenic mice (n = 5 per group) were prime-vaccinated with rVSV Ind -msp-S F -Gtc and boost vaccinated with rVSV Ind -msp-S F -Gtc or rVSV NJ -msp-S F -Gtc two weeks after prime-vaccination. Four weeks after boost-vaccination , mice were challenged intranasally with 1x10 5 PFU of SARS-CoV-2. The survival and body weight of each mouse was monitored daily. (A) Average bodyweights of mice in each vaccinated group. (B) Individual body weights for mice vaccinated with rVSV-Mock and challenged with SARS-CoV-2. (C) Mouse survival after SARS-CoV-2 challenge. VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Right lobes of mice lungs were aseptically removed from the mice on day 3, day 7, and day 15 after SARS-CoV-2 challenge. Infectious SARS-CoV-2 was quantified by plaque assay on Vero E6 cells. Statistical significance was determined by two-way ANOVA with Tukey’s correction (****, p< 0.0001). VSV-Mock denotes VSV vector alone without any gene insert.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Plaque Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2

doi: 10.1371/journal.ppat.1010092

Figure Lengend Snippet: Human ACE2 transgenic mice were vaccinated and challenged with SARS-CoV-2 as described in . Left lobes of mice lungs were fixed in 10% buffered formalin on day 3 and day 7 after the SARS-CoV-2 challenge. Lung tissues were processed and embedded in low-melting paraffin, sectioned to a thickness of 3 μm, and stained with hematoxylin and eosin. Stained tissues were examined under a light microscope (Olympus CS41, Japan) with 100X magnification. Note: a, alveolus; b, bronchiole; v, blood vessels. (A) Lung tissue 3 days after the SARS-CoV-2 challenge. (B) Lung tissue 7 days after SARS-CoV-2 challenge. Arrows show infiltration of inflammatory cells (lymphocytes and macrophages). G1: empty vector infected mice, G2: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G3: 5X10 8 of rVSV Ind -msp-S F -Gtc/ rVSV Ind -msp-S F -Gtc vaccinated mice, G4: 5X10 7 of rVSV Ind -msp-S F -Gtc/ rVSV NJ -msp-S F -Gtc vaccinated mice, G5: uninfected mice.

Article Snippet: S2 protein was detected by another rabbit antibody against SARS-CoV-2 S2 (Sino Biological, cat# 40590-T62).

Techniques: Transgenic Assay, Staining, Light Microscopy, Plasmid Preparation, Infection

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Production and characterization of the anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Expression of biotin peptide-6 × His-tagged SARS-CoV-2 N protein (SARS-CoV-2 N-Bio-His 6 ). Recombinant SARS-CoV-2 N-Bio-His 6 protein was expressed in ExpiCHO cells and purified from cell culture supernatants using Ni-NTA agarose chromatography. The purified recombinant protein was analyzed by SDS-PAGE (left) and western blotting with peroxidase-conjugated streptavidin (middle) and anti-His-tag antibody (right). Arrowhead, biotin peptide-6 × His-tagged SARS-CoV-2 N protein; R, reducing condition; NR, non-reducing condition. (B) Expression of biotin peptide-6 × His-tagged MERS-CoV N protein (MERS-CoV N-Bio-His 6 ). The purified recombinant protein was analyzed by SDS-PAGE. Arrowhead, biotin peptide-6 × His-tagged MERS-CoV N protein. (C) The recombinant SARS-CoV-2 N-Bio-His 6 protein and CpG-DNA were combined in a DOPE:CHEMS complex and the complex was injected intraperitoneally into BALB/c mice ( n = 4) three times. ELISA was performed with mouse sera to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (D) Ascites were collected from mice injected with cloned hybridoma cells (1G10C4). ELISA was performed with the ascites to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (E) The monoclonal antibody was purified from the ascitic fluid using Protein-A column chromatography and analyzed using SDS-PAGE. HC, heavy chain; LC, light chain. (F) Subclasses of the monoclonal antibody were identified by ELISA. (G) The detection limit of the monoclonal antibody against SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA. (H) Binding of the monoclonal antibody to recombinant SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Chromatography, SDS Page, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Clone Assay, Column Chromatography, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Specificity of the anti-SARS-CoV-2 N protein monoclonal antibody. (A) Analysis of monoclonal antibody specificity. Recombinant SARS-CoV-2 N-Bio-His 6 protein or recombinant MERS-CoV N-Bio-His 6 protein was captured on streptavidin-coated 96-well immunoplates and then incubated with anti-SARS-CoV-2 N protein monoclonal antibody. Reactivity of the monoclonal antibody to each recombinant protein was determined by ELISA. (B) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoblotted with the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Immunoprecipitation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A,B) Western blot analyses for the detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells (A) and Calu-3 cells (B) were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 72 h. Cell lysates were analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) Confocal microscopy was used to detect N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells were cultured on cover glass in 12-well plates and infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 48 h. The infected Vero cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with anti-SARS-CoV-2 N protein-specific monoclonal antibody or normal IgG and then with Alexa Fluor 488–conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst 33258. Images were obtained by confocal microscope. Scale bar, 10 μm.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Infection, Western Blot, Confocal Microscopy, Cell Culture, Incubation, Staining, Microscopy

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in SARS-CoV-2 S, GH, or GR clade virus particles with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Western blot analyses. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Virus titers were measured by plaque assay. (B) Immunoprecipitation analysis. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Western Blot, Infection, Cell Culture, Lysis, Plaque Assay, Immunoprecipitation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: N protein in SARS-CoV-2 S, GH, or GR clade virus particles interact with SARS-CoV-2 Spike CD. (A) Expression of SARS-CoV-2 Spike CD-Fc. The recombinant Fc control protein and the SARS-CoV-2 Spike CD-Fc fusion protein were expressed in ExpiCHO cells, purified from cell culture supernatants using Protein A column chromatography and analyzed by SDS-PAGE and Coomassie blue staining. (B) Interaction of the N protein in virus particles with SARS-CoV-2 Spike CD. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. Cell culture supernatants were lysed with cell lysis buffer and incubated with Fc or SARS-CoV-2 Spike CD-Fc. Fc-bound proteins were pulled down with Protein A beads and subjected to western blot analysis using the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Western blot of virus lysates is shown as a control.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Column Chromatography, SDS Page, Staining, Infection, Lysis, Incubation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant proteins. (A) Schematic of the bait and prey assay system. (B) Immunoplates (96-well) were coated with streptavidin and then recombinant SARS-CoV-2 N-Bio-His 6 protein was added to each well. After addition of MERS-CoV Spike CD-Fc or SARS-CoV-2 Spike CD-Fc, the amount of CoV Spike CD-human Fc fusion protein bound to recombinant SARS-CoV-2 N-Bio-His 6 protein in the wells was determined by ELISA. (C) Specificity of the interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant protein. Serially diluted non-biotinylated-recombinant SARS-CoV-2 N-His 6 protein was incubated with SARS-CoV-2 Spike CD-Fc and then added to the wells containing SARS-CoV-2 N-Bio-His 6 -coated streptavidin. The extent of the competition was measured by ELISA using HRP-conjugated anti-human IgG Fc antibody.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of SARS-CoV-2 in cell culture media using SARS-CoV-2 N protein-specific monoclonal antibody and recombinant SARS-CoV-2 Spike CD-Fc protein. (A) Schematic of the ELISA. (B) SARS-CoV-2 clade S and HCoV-OC43 and (C) SARS-CoV-2 clade GH and clade GR in cell culture supernatants were lysed with cell lysis buffer and serially diluted in PBST. These virus lysates were added into 96-well immunoplates coated with SARS-CoV-2 N protein-specific monoclonal antibody. After incubation for 2 h at room temperature, recombinant SARS-CoV-2 Spike CD-Fc protein was added, and then HRP-conjugated anti-human IgG Fc antibody was added to each well. The amount of SARS-CoV-2 N protein in each well was determined by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Incubation

Journal: Viruses

Article Title: Characterization of Immune Response Diversity in Rodents Vaccinated with a Vesicular Stomatitis Virus Vectored COVID-19 Vaccine

doi: 10.3390/v14061127

Figure Lengend Snippet: Characterization of the recombinant VSV viruses. ( a ) Schematic diagrams showing genome organization of the rVSV. VSV g was replaced by SARS-CoV-2 S to generate rVSV-ΔG-S and rVSV-ΔG-S-eGFP. ( b ) Indirect immunofluorescence identification results of rVSV-ΔG-S and rVSV-ΔG-S-eGFP. Vero E6 cells were infected with rVSV-ΔG-S or rVSV-ΔG-S-eGFP and recognized by SARS-CoV-2-specific antibody and Alexa Fluor 568-conjugated secondary antibody, presented red fluorescence, rVSV-ΔG-S-eGFP presented green fluorescence. No fluorescence signal was observed in the VSV control. ( c ) Lysates of rVSV-ΔG-S/rVSV-ΔG-S-eGFP-infected Vero E6 cells, and VSV-infected Vero cells were blotted with an antibody recognizing SARS-CoV-2 S protein, a band representing S at 190 kDa was detected in rVSV-ΔG-S and rVSV-ΔG-S-eGFP group, no band was observed in the VSV control. ( d ) Growth kinetics of rVSV-ΔG-S, rVSV-ΔG-S-eGFP and VSV. Vero E6 cells were infected with the recombinant VSV viruses (MOI = 0.01) and virus titers in the supernatant were measured at the indicated time points post-infection.

Article Snippet: Rabbit polyclonal antibody against SARS-CoV-2 S protein (Cat. 40589-T62) was purchased from Sino Biological Inc (Beijing, China).

Techniques: Recombinant, Immunofluorescence, Infection, Fluorescence

Journal: Viruses

Article Title: Characterization of Immune Response Diversity in Rodents Vaccinated with a Vesicular Stomatitis Virus Vectored COVID-19 Vaccine

doi: 10.3390/v14061127

Figure Lengend Snippet: Virus-specific antibody and neutralizing antibody induced by rVSVΔG-S. ( a ) Weight change monitoring of BALB/c mice following vaccination for 7 days. i.m., i.n. and control refer to i.m. ( n = 10), i.n. inoculation group ( n = 10) and PBS inoculation control group ( n = 10), respectively same in ( b – f ). ( b ) RBD-specific IgG in mouse serum samples at 14, 28 and 42 dpv. ( c ) nAbs in the serum of mice at 14, 28, 42 and 56 dpv. ( d ) Weight changes in golden hamsters 7 days after vaccination. ( e ) RBD-specific IgG in the serum of golden hamsters at 14 and 28 dpv. ( f ) nAb titers in hamster serum samples at 14, 28 dpv and post-SARS-CoV-2 challenge. Data are presented as mean ± SEM. (* p < 0.05, ** p < 0.01,*** p < 0.001, **** p < 0.0001).

Article Snippet: Rabbit polyclonal antibody against SARS-CoV-2 S protein (Cat. 40589-T62) was purchased from Sino Biological Inc (Beijing, China).

Techniques:

Journal: Viruses

Article Title: Characterization of Immune Response Diversity in Rodents Vaccinated with a Vesicular Stomatitis Virus Vectored COVID-19 Vaccine

doi: 10.3390/v14061127

Figure Lengend Snippet: Antibody subtype and cytokines induced by rVSVΔG-S in BALB/c mice. I.m., i.n. and control refer to i.m. ( n = 4), i.n. inoculation group ( n = 4) and PBS inoculation control group ( n = 2), respectively (same in b–f). ( a ) RBD-specific IgG1 and IgG2a induced by the rVSV vaccine in mice. ( b , c ) Splenocyte IFN-γ and IL-4 induced by the rVSV vaccine in mice. ( d ) Ratio of IgG2a/IgG1 in i.m. and i.n. group. ( e , f ) CD3+CD4+ and CD3+CD8+ positive T cell proportions in splenocyte before and post-stimulation with 20 μg/mL RBD protein of SARS-CoV-2 for 36 h at 37 °C, 5% CO 2 . Data are presented as mean ± SEM. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Rabbit polyclonal antibody against SARS-CoV-2 S protein (Cat. 40589-T62) was purchased from Sino Biological Inc (Beijing, China).

Techniques:

Journal: Viruses

Article Title: Characterization of Immune Response Diversity in Rodents Vaccinated with a Vesicular Stomatitis Virus Vectored COVID-19 Vaccine

doi: 10.3390/v14061127

Figure Lengend Snippet: Protective efficacy of rVSV-ΔG-S in golden hamsters following SARS-CoV-2 challenge. i.m., i.n. and control refer to i.m. ( n = 5), i.n. inoculation group ( n = 5) and PBS inoculation group ( n = 2) who were challenged with SARS-CoV-2, respectively (same in b–f). Unchallenged control refers to hamsters inoculated with PBS and free from SARS-CoV-2 challenge ( n = 6). ( a ) Survival rate following SARS-CoV-2 challenge. ( b ) Body weight change in golden hamsters following SARS-CoV-2 challenge. ( c , d ) SARS-CoV-2 viral RNA copies in the lungs and nasal turbinates following SARS-CoV-2 challenge. ( e , f ) SARS-CoV-2 viral titers in the lungs and nasal turbinates following SARS-CoV-2 challenge. Data are presented as mean ± SEM. (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Article Snippet: Rabbit polyclonal antibody against SARS-CoV-2 S protein (Cat. 40589-T62) was purchased from Sino Biological Inc (Beijing, China).

Techniques:

Journal: Viruses

Article Title: Characterization of Immune Response Diversity in Rodents Vaccinated with a Vesicular Stomatitis Virus Vectored COVID-19 Vaccine

doi: 10.3390/v14061127

Figure Lengend Snippet: Representative H&E staining and IHC of lung sections from SARS-CoV-2-infected golden hamsters at 3 days post-infection. Mock refers to hamsters inoculated with PBS and free from SARS-CoV-2 challenged. i.m., i.n. and control refer to i.m. inoculation group, i.n. inoculation group and PBS inoculation group who were challenged with SARS-CoV-2, respectively. In H&E staining sections, the thickened alveolar wall and proliferated epithelial cells are marked with yellow arrows, broken alveolar diaphragms and atrophic alveoli are marked with red arrows. In IHC sections, SARS-CoV-2 antigens that were recognized by SARS-CoV-2-N-specific antibodies were marked with red arrows.

Article Snippet: Rabbit polyclonal antibody against SARS-CoV-2 S protein (Cat. 40589-T62) was purchased from Sino Biological Inc (Beijing, China).

Techniques: Staining, Infection

Journal: Nature Communications

Article Title: Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters

doi: 10.1038/s41467-024-48414-x

Figure Lengend Snippet: a – f Detection of (ancestral) spike (S)-specific binding IgG antibodies ( a , d ), ancestral SARS-CoV-2 neutralizing antibodies ( b , e ), and T-cell responses measured by interferon-gamma (IFN-γ) release assay (IGRA) ( c , f ) after Omicron BA.1 ( a – c ) or BA.5 ( d – f ) bivalent booster vaccination at baseline, and 7 days, 28 days, and 3 months post-boost. Colors indicate the specific prime-boost regimen (orange = Ad26.COV2.S prime, BNT162b2 Omicron BA.1 or BA.5 boost; yellow = Ad26.COV2.S prime, mRNA-1273.214 or mRNA-1273.222 boost; dark red = SARS-CoV-2 infection prime, BNT162b2 Omicron BA.1 or BA.5 boost; light red = SARS-CoV-2 infection prime, mRNA-1273.214 or mRNA-1273.222 boost; dark blue = mRNA-based prime, BNT162b2 Omicron BA.1 or BA.5 boost; light blue = mRNA-based prime, mRNA-1273.214 or mRNA-1273.222 boost). Data are shown in box-and-whisker plots, with the horizontal lines indicating the median, the bounds of the boxes indicating the interquartile range (IQR), and the whiskers indicating the range. Bold numbers above the plots represent the respective geometric mean (titer) per timepoint. The line graphs next to each panel depict a time course of the respective geometric mean values with 95% confidence intervals. The number of biologically independent samples (serum or whole blood) used per assay is shown in Supplementary Table .

Article Snippet: Four hundred PFU of either SARS-CoV-2 variant in an equal volume of OptiMEM medium were added to the diluted sera and incubated at 37 °C for 1 h. The antibody-virus mix was then transferred to Calu-3 cells and incubated at 37 °C for 8 h. Afterwards, the cells were fixed in 10% neutral-buffered formalin, permeabilized in 70% ethanol, and the plaques stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (1:1,000; Sino Biological, #40143-T62) and a secondary peroxidase-labeled goat-anti-rabbit IgG antibody (1:2000; Dako, #P0448).

Techniques: Binding Assay, Release Assay, Infection, Whisker Assay

Journal: Nature Communications

Article Title: Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters

doi: 10.1038/s41467-024-48414-x

Figure Lengend Snippet: a – f Detection of (ancestral) spike (S)-specific binding IgG antibodies ( a , b ), ancestral SARS-CoV-2 neutralizing antibodies ( c , d ), and T-cell responses measured by interferon-gamma (IFN-γ) release assay (IGRA) ( e , f ) based on the different combinations of original priming regimen after Omicron BA.1 ( a , c , e ) or BA.5 ( b , d , f ) bivalent booster vaccination at baseline, and 7 days, 28 days, and 3 months post-boost. Colors indicate the specific prime-boost regimen (orange = Ad26.COV2.S prime, BNT162b2 Omicron BA.1 or BA.5 boost; yellow = Ad26.COV2.S prime, mRNA-1273.214 or mRNA-1273.222 boost; dark blue = mRNA-based prime, BNT162b2 Omicron BA.1 or BA.5 boost; light blue = mRNA-based prime, mRNA-1273.214 or mRNA-1273.222 boost). Data are shown in box-and-whisker plots, with the horizontal lines indicating the median, the bounds of the boxes indicating the interquartile range (IQR), and the whiskers indicating the range. Bold numbers above the plots represent the respective geometric mean (titer) per timepoint. The line graphs next to each panel depict a time course of the respective geometric mean values with 95% confidence intervals. The number of biologically independent samples (serum or whole blood) used per assay is shown in Supplementary Table .

Article Snippet: Four hundred PFU of either SARS-CoV-2 variant in an equal volume of OptiMEM medium were added to the diluted sera and incubated at 37 °C for 1 h. The antibody-virus mix was then transferred to Calu-3 cells and incubated at 37 °C for 8 h. Afterwards, the cells were fixed in 10% neutral-buffered formalin, permeabilized in 70% ethanol, and the plaques stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (1:1,000; Sino Biological, #40143-T62) and a secondary peroxidase-labeled goat-anti-rabbit IgG antibody (1:2000; Dako, #P0448).

Techniques: Binding Assay, Release Assay, Whisker Assay

Journal: Nature Communications

Article Title: Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters

doi: 10.1038/s41467-024-48414-x

Figure Lengend Snippet: a – f Detection of (ancestral) spike (S)-specific binding IgG antibodies ( a , d ), ancestral SARS-CoV-2 neutralizing antibodies ( b , e ), and T-cell responses measured by interferon-gamma (IFN-γ) release assay (IGRA) ( c , f ) after Omicron BA.1 ( a – c ) or BA.5 ( d – f ) bivalent booster vaccination with either BNT162b2 Omicron BA.1 or BA.5 (blue) or mRNA-1273.214 or mRNA-1273.222 (green) at baseline, and 7 days, 28 days, and 3 months post-boost. Data are shown in box-and-whisker plots, with the horizontal lines indicating the median, the bounds of the boxes indicating the interquartile range (IQR), and the whiskers indicating the range. Bold numbers above the plots represent the respective geometric mean (titer) per timepoint. Italic numbers below the plots indicate fold changes relative to the baseline. The number of biologically independent samples (serum or whole blood) used per assay is shown in Supplementary Table .

Article Snippet: Four hundred PFU of either SARS-CoV-2 variant in an equal volume of OptiMEM medium were added to the diluted sera and incubated at 37 °C for 1 h. The antibody-virus mix was then transferred to Calu-3 cells and incubated at 37 °C for 8 h. Afterwards, the cells were fixed in 10% neutral-buffered formalin, permeabilized in 70% ethanol, and the plaques stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (1:1,000; Sino Biological, #40143-T62) and a secondary peroxidase-labeled goat-anti-rabbit IgG antibody (1:2000; Dako, #P0448).

Techniques: Binding Assay, Release Assay, Whisker Assay

Journal: Nature Communications

Article Title: Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters

doi: 10.1038/s41467-024-48414-x

Figure Lengend Snippet: a , b Detection of neutralizing antibodies targeting ancestral SARS-CoV-2 and Omicron BA.1, BA.5, and XBB.1.5 variants after Omicron BA.1 ( a ) or BA.5 ( b ) bivalent booster vaccination at baseline, and 7 days, 28 days, and 3 months post-boost. Colors indicate the specific prime-boost regimen (orange = Ad26.COV2.S prime, BNT162b2 Omicron BA.1 or BA.5 boost; yellow = Ad26.COV2.S prime, mRNA-1273.214 or mRNA-1273.222 boost; dark red = SARS-CoV-2 infection prime, BNT162b2 Omicron BA.1 or BA.5 boost; light red = SARS-CoV-2 infection prime, mRNA-1273.214 or mRNA-1273.222 boost; dark blue = mRNA-based prime, BNT162b2 Omicron BA.1 or BA.5 boost; light blue = mRNA-based prime, mRNA-1273.214 or mRNA-1273.222 boost). c – f Correlation between 50% plaque reduction neutralization (PRNT 50 ) titers against ancestral SARS-CoV-2 and the Omicron BA.1 ( c , d ) or BA.5 ( e , f ) variants over time after Omicron BA.1 ( c , e ) or BA.5 ( d , f ) vaccination at baseline, and 7 days, 28 days, and 3 months post-boost. Colored symbols indicate the specific timepoints (yellow = baseline [0 d]; teal = 7 d; purple = 28 d; orange = 77 d [c,e]/98 d [ d , f ]). The arrows connect the correlated geometric means (+95% confidence intervals [CI]) per timepoint and visualize the neutralization kinetics. g , h Radar plots depicting the variant-specific PRNT 50 titers relative to ancestral SARS-CoV-2 neutralization (set to 100%) after vaccination with bivalent Omicron BA.1 or BA.5. The plots are grouped either by the administered Omicron BA.1 (orange) or BA.5 (purple) bivalent booster vaccination ( g ) or the original priming regimen (vector-based = yellow; mRNA-based = blue) after Omicron BA.5 bivalent vaccination ( h ). Data in ( a , b ) are shown in box-and-whisker plots, with the horizontal lines indicating the median, the bounds of the boxes indicating the interquartile range (IQR), and the whiskers indicating the range. Bold numbers above the plots represent the respective geometric mean (titer) per timepoint. The line graphs next to each panel depict a time course of the respective geometric mean values with 95% confidence intervals. The number of biologically independent sera is shown in Supplementary Table .

Article Snippet: Four hundred PFU of either SARS-CoV-2 variant in an equal volume of OptiMEM medium were added to the diluted sera and incubated at 37 °C for 1 h. The antibody-virus mix was then transferred to Calu-3 cells and incubated at 37 °C for 8 h. Afterwards, the cells were fixed in 10% neutral-buffered formalin, permeabilized in 70% ethanol, and the plaques stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (1:1,000; Sino Biological, #40143-T62) and a secondary peroxidase-labeled goat-anti-rabbit IgG antibody (1:2000; Dako, #P0448).

Techniques: Infection, Neutralization, Variant Assay, Plasmid Preparation, Whisker Assay

Journal: Nature Communications

Article Title: Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters

doi: 10.1038/s41467-024-48414-x

Figure Lengend Snippet: a Sampling procedure for participants in the postponed boost group who had a breakthrough infection before their intended vaccination with the bivalent Omicron BA.5 booster vaccine ( n = 12). They were subsequently excluded from the vaccination trajectory and invited to participate in a sub-study on the immunogenicity of natural SARS-CoV-2 infection. Samples were collected at enrollment, and 7 and 28 days after the participants tested positive. Created with BioRender.com. b – d Detection of (ancestral) S-specific binding IgG antibodies ( b ), T-cell responses measured by interferon-gamma (IFN-γ) release assay (IGRA) ( c ), and neutralizing antibodies targeting ancestral SARS-CoV-2 and Omicron BA.1, BA.5, and XBB.1.5 variants ( d ) before, and 7 and 28 days after breakthrough infection, which was contracted before intended vaccination with the bivalent Omicron BA.5 booster vaccine (yellow = Ad26.COV2.S prime; red = SARS-CoV-2 infection prime; blue = mRNA-based prime). Data are shown in box-and-whisker plots, with the horizontal lines indicating the median, the bounds of the boxes indicating the interquartile ranges (IQR), and the whiskers indicating the range. Bold numbers above the plots represent the respective geometric mean (titer) per timepoint. The line graphs next to each panel depict a time course of the respective geometric mean values with 95% confidence intervals. While the solid lines show the geometric mean values of the data from the box-and-whisker plots in the same panel, the dashed lines show reference values from comparable timepoints after either Omicron BA.1 (green) or BA.5 (orange) bivalent vaccination.

Article Snippet: Four hundred PFU of either SARS-CoV-2 variant in an equal volume of OptiMEM medium were added to the diluted sera and incubated at 37 °C for 1 h. The antibody-virus mix was then transferred to Calu-3 cells and incubated at 37 °C for 8 h. Afterwards, the cells were fixed in 10% neutral-buffered formalin, permeabilized in 70% ethanol, and the plaques stained with a polyclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (1:1,000; Sino Biological, #40143-T62) and a secondary peroxidase-labeled goat-anti-rabbit IgG antibody (1:2000; Dako, #P0448).

Techniques: Sampling, Infection, Binding Assay, Release Assay, Whisker Assay