rabbit polyclonal anti mers cov spike protein antibody  (Sino Biological)


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    Name:
    MERS CoV Spike Protein Antibody Rabbit PAb
    Description:
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 Catalog 40069 V08B AFS88936 1 Met1 Trp1297 MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 specific IgG was purified by MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 affinity chromatography
    Catalog Number:
    40069-T30
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    MERS CoV
    Applications:
    ELISA
    Immunogen:
    Recombinant MERS-CoV (NCoV / Novel coronavirus) Spike Protein (ECD, aa 1-1297) Protein (Catalog#40069-V08B)
    Product Aliases:
    Anti-coronavirus s1 Antibody, Anti-coronavirus s2 Antibody, Anti-coronavirus spike Antibody, Anti-cov spike Antibody, Anti-ncov RBD Antibody, Anti-ncov s1 Antibody, Anti-ncov s2 Antibody, Anti-ncov spike Antibody, Anti-RBD Antibody, Anti-S Antibody, Anti-s1 Antibody, Anti-Spike RBD Antibody
    Antibody Type:
    PAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological rabbit polyclonal anti mers cov spike protein antibody
    Neutralizing activity of mAbs specific for S1 and the RBD of the S protein. Neutralization potency of RBD and S1-specific mAbs evaluated using a <t>MERS-CoV</t> EMC pseudovirus-neutralization assay (A) and a live-virus PRNT assay (B). The percent inhibition by the mAbs was determined by comparison with untreated cells. Each experiment was performed in triplicate, and data represent the mean of each experiment with standard errors.
    Produced in rabbits immunized with purified recombinant MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 Catalog 40069 V08B AFS88936 1 Met1 Trp1297 MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 specific IgG was purified by MERS CoV NCoV Novel coronavirus Spike Protein ECD aa 1 1297 affinity chromatography
    https://www.bioz.com/result/rabbit polyclonal anti mers cov spike protein antibody/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti mers cov spike protein antibody - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusion-stabilized spike protein of Middle East respiratory syndrome coronavirus"

    Article Title: Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusion-stabilized spike protein of Middle East respiratory syndrome coronavirus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0232757

    Neutralizing activity of mAbs specific for S1 and the RBD of the S protein. Neutralization potency of RBD and S1-specific mAbs evaluated using a MERS-CoV EMC pseudovirus-neutralization assay (A) and a live-virus PRNT assay (B). The percent inhibition by the mAbs was determined by comparison with untreated cells. Each experiment was performed in triplicate, and data represent the mean of each experiment with standard errors.
    Figure Legend Snippet: Neutralizing activity of mAbs specific for S1 and the RBD of the S protein. Neutralization potency of RBD and S1-specific mAbs evaluated using a MERS-CoV EMC pseudovirus-neutralization assay (A) and a live-virus PRNT assay (B). The percent inhibition by the mAbs was determined by comparison with untreated cells. Each experiment was performed in triplicate, and data represent the mean of each experiment with standard errors.

    Techniques Used: Activity Assay, Neutralization, Plaque Reduction Neutralization Test, Inhibition

    Binding of human mAbs to S1 and RBD of the MERS-CoV S protein. Binding specificity of the mAbs was assessed by ELISA with a soluble S-2P trimer, S1, RBD sub-domains, and S2-mFc. Among the 11 purified mAbs, six were specific to RBD and five were specific to S1 (non-RBD). RBD-specific mAbs bound to both RBD and S1 protein. No S2-specific mAbs (blue line) were identified. Data points represent the mean of three technical replicates with standard errors.
    Figure Legend Snippet: Binding of human mAbs to S1 and RBD of the MERS-CoV S protein. Binding specificity of the mAbs was assessed by ELISA with a soluble S-2P trimer, S1, RBD sub-domains, and S2-mFc. Among the 11 purified mAbs, six were specific to RBD and five were specific to S1 (non-RBD). RBD-specific mAbs bound to both RBD and S1 protein. No S2-specific mAbs (blue line) were identified. Data points represent the mean of three technical replicates with standard errors.

    Techniques Used: Binding Assay, Protein Binding, Enzyme-linked Immunosorbent Assay, Purification

    MERS-CoV RBD-specific mAb inhibits viral replication in mouse tissues. Four mice were treated with the KNIH90-F1 mAb (8 mg/kg) 24 h after MERS-CoV KNIH-002 strain lethal challenge (1 × 10 5 pfu/mouse) under therapeutic conditions. Moreover, four mice were infected with MERS-CoV 24 h after administration of the same amount of mAb under prophylactic conditions. At 4-days post-challenge, all mice were humanely sacrificed, and tissues (lung and brain) were collected for viral titer analysis by plaque assays (A) and real-time RT-PCR (B). Viral genome copies in tissues were determined by real-time RT-PCR targeting the Orf1a gene. Data represent the mean ± standard deviation. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005.
    Figure Legend Snippet: MERS-CoV RBD-specific mAb inhibits viral replication in mouse tissues. Four mice were treated with the KNIH90-F1 mAb (8 mg/kg) 24 h after MERS-CoV KNIH-002 strain lethal challenge (1 × 10 5 pfu/mouse) under therapeutic conditions. Moreover, four mice were infected with MERS-CoV 24 h after administration of the same amount of mAb under prophylactic conditions. At 4-days post-challenge, all mice were humanely sacrificed, and tissues (lung and brain) were collected for viral titer analysis by plaque assays (A) and real-time RT-PCR (B). Viral genome copies in tissues were determined by real-time RT-PCR targeting the Orf1a gene. Data represent the mean ± standard deviation. * P ≤ 0.05; ** P ≤ 0.005; *** P ≤ 0.0005.

    Techniques Used: Mouse Assay, Infection, Quantitative RT-PCR, Standard Deviation

    Histopathological changes of the lungs and brains in MERS-CoV-infected mice. Six mice were treated with the KNIH90-F1 mAb (8 mg/kg) 24 h after lethal challenge. At 4- and 7-days post-challenge, all mice were sacrificed, and lungs (A) and brains (B) were collected. Tissue sections were stained with H E (magnification: 400×).
    Figure Legend Snippet: Histopathological changes of the lungs and brains in MERS-CoV-infected mice. Six mice were treated with the KNIH90-F1 mAb (8 mg/kg) 24 h after lethal challenge. At 4- and 7-days post-challenge, all mice were sacrificed, and lungs (A) and brains (B) were collected. Tissue sections were stained with H E (magnification: 400×).

    Techniques Used: Infection, Mouse Assay, Staining

    MERS-CoV RBD-specific mAb protects mice from lethal challenge. Five hDPP4 TG mice were intraperitoneally administered different doses of KNIH90-F1 24 h after intranasal inoculation with the MERS-CoV KNIH-002 strain (1 × 10 5 pfu/30 μl). Body weight change (A) and survival (B) were monitored for 14 days.
    Figure Legend Snippet: MERS-CoV RBD-specific mAb protects mice from lethal challenge. Five hDPP4 TG mice were intraperitoneally administered different doses of KNIH90-F1 24 h after intranasal inoculation with the MERS-CoV KNIH-002 strain (1 × 10 5 pfu/30 μl). Body weight change (A) and survival (B) were monitored for 14 days.

    Techniques Used: Mouse Assay

    2) Product Images from "CD26/DPP4 Cell-Surface Expression in Bat Cells Correlates with Bat Cell Susceptibility to Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection and Evolution of Persistent Infection"

    Article Title: CD26/DPP4 Cell-Surface Expression in Bat Cells Correlates with Bat Cell Susceptibility to Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection and Evolution of Persistent Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112060

    Expression of human CD26/DPP4 confers MERS-CoV susceptibility to otherwise resistant bat cells. (A) Viral yields from MERS-CoV-resistant PESU-B5L, R05T, R06E, and Tb1Lu bat cells. Cells were transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid and exposed 48 h later to MERS-CoV/EMC at an MOI of 3. Supernatants were harvested at 24 h after virus exposure for quantification of virus yields by plaque assay. (B) Same experiment: representative immunofluorescence assay (IFA) images of cells stained with anti-MERS-CoV spike protein antibody (green, top) or anti-human CD26/DPP4 antibody (red, bottom). (C) Merged IFA images demonstrate colocalization of MERS-CoV spike protein and CD26/DPP4. (D). Viral yields from MERS-CoV-susceptible bat cells transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid using procedures identical to resistant cells in (A) except that cells were exposed to virus 24 h after transfection. Error bars indicate the standard deviation of duplicate samples.
    Figure Legend Snippet: Expression of human CD26/DPP4 confers MERS-CoV susceptibility to otherwise resistant bat cells. (A) Viral yields from MERS-CoV-resistant PESU-B5L, R05T, R06E, and Tb1Lu bat cells. Cells were transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid and exposed 48 h later to MERS-CoV/EMC at an MOI of 3. Supernatants were harvested at 24 h after virus exposure for quantification of virus yields by plaque assay. (B) Same experiment: representative immunofluorescence assay (IFA) images of cells stained with anti-MERS-CoV spike protein antibody (green, top) or anti-human CD26/DPP4 antibody (red, bottom). (C) Merged IFA images demonstrate colocalization of MERS-CoV spike protein and CD26/DPP4. (D). Viral yields from MERS-CoV-susceptible bat cells transfected with a plasmid expressing human CD26/DPP4 or empty control plasmid using procedures identical to resistant cells in (A) except that cells were exposed to virus 24 h after transfection. Error bars indicate the standard deviation of duplicate samples.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Plaque Assay, Immunofluorescence, Staining, Standard Deviation

    Six of ten tested bat cell lines are susceptible to MERS-CoV infection. (A and B) Ten different bat cell lines were exposed to MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. Supernatants were harvested at days 0, 1, 3, and 5 after virus exposure, and virus yields were determined by plaque assay on Vero cells. Error bars indicate the standard deviation of triplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cell lines exposed to MERS-CoV/EMC (C) or MERS-CoV/Jor. (D) 1 (D1) or 3 (D3) days after virus exposure and stained against MERS-CoV spike protein (green). (E) Same experiment: TEM images of bat cells infected with MERS-CoV/EMC at day 1 after virus exposure. Red arrows point at double-membrane vesicles (DMVs) typical of coronavirus infections.
    Figure Legend Snippet: Six of ten tested bat cell lines are susceptible to MERS-CoV infection. (A and B) Ten different bat cell lines were exposed to MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. Supernatants were harvested at days 0, 1, 3, and 5 after virus exposure, and virus yields were determined by plaque assay on Vero cells. Error bars indicate the standard deviation of triplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cell lines exposed to MERS-CoV/EMC (C) or MERS-CoV/Jor. (D) 1 (D1) or 3 (D3) days after virus exposure and stained against MERS-CoV spike protein (green). (E) Same experiment: TEM images of bat cells infected with MERS-CoV/EMC at day 1 after virus exposure. Red arrows point at double-membrane vesicles (DMVs) typical of coronavirus infections.

    Techniques Used: Infection, Plaque Assay, Standard Deviation, Immunofluorescence, Staining, Transmission Electron Microscopy

    Persistent MERS-CoV infection of bat cells induces downregulation of bat cell CD26/DPP4 expression. Bat cell lines susceptible to infection were infected with MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. After 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1∶10 dilution in new flasks. Subsequently, the persistently infected cells were passaged at a 1∶10 dilution weekly. Error bars indicate the standard deviation of duplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cells persistently infected with MERS-CoV/EMC (C) or MERS-CoV/Jor (D) at day +33 stained with anti-MERS-CoV spike protein antibody (green). (E) Same experiment: TEM images of bat cells persistently infected with MERS-CoV/EMC at day 56. (F) Flow cytometry data of CD26/DPP4 surface expression (red line: anti-human CD26-/DPP4 antibody; black line: control antibody) in persistently infected cells. (G) CD26/DPP4 expression in persistently infected EidNi/41.3 cells (day 63) as detected by western blot.
    Figure Legend Snippet: Persistent MERS-CoV infection of bat cells induces downregulation of bat cell CD26/DPP4 expression. Bat cell lines susceptible to infection were infected with MERS-CoV/EMC (A) or MERS-CoV/Jor (B) at an MOI of 1. After 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1∶10 dilution in new flasks. Subsequently, the persistently infected cells were passaged at a 1∶10 dilution weekly. Error bars indicate the standard deviation of duplicate samples. (C and D) Same experiment: immunofluorescence assay (IFA) images of bat cells persistently infected with MERS-CoV/EMC (C) or MERS-CoV/Jor (D) at day +33 stained with anti-MERS-CoV spike protein antibody (green). (E) Same experiment: TEM images of bat cells persistently infected with MERS-CoV/EMC at day 56. (F) Flow cytometry data of CD26/DPP4 surface expression (red line: anti-human CD26-/DPP4 antibody; black line: control antibody) in persistently infected cells. (G) CD26/DPP4 expression in persistently infected EidNi/41.3 cells (day 63) as detected by western blot.

    Techniques Used: Infection, Expressing, Plaque Assay, Standard Deviation, Immunofluorescence, Staining, Transmission Electron Microscopy, Flow Cytometry, Western Blot

    Anti-human CD26/DPP4 antibody inhibits MERS-CoV infection of bat cells. RoNi/7.1 or Huh-7 cells (control) were treated with increasing concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml) of anti-human CD26/DPP4 antibody or control antibody and then exposed to MERS-CoV/EMC at an MOI of 1. (A) After 24 h, viral yields in supernatants were determined by plaque assay. (B) Cellular infection was determined by immunofluorescence assay (IFA) with an anti-MERS-CoV spike protein antibody (green). (B left) The percentage of infected cells was analyzed by high content imaging. (B right) Representative IFA images. Error bars indicate the standard deviation of triplicate samples.
    Figure Legend Snippet: Anti-human CD26/DPP4 antibody inhibits MERS-CoV infection of bat cells. RoNi/7.1 or Huh-7 cells (control) were treated with increasing concentrations (0, 1.25, 2.5, 5, 10, and 20 µg/ml) of anti-human CD26/DPP4 antibody or control antibody and then exposed to MERS-CoV/EMC at an MOI of 1. (A) After 24 h, viral yields in supernatants were determined by plaque assay. (B) Cellular infection was determined by immunofluorescence assay (IFA) with an anti-MERS-CoV spike protein antibody (green). (B left) The percentage of infected cells was analyzed by high content imaging. (B right) Representative IFA images. Error bars indicate the standard deviation of triplicate samples.

    Techniques Used: Infection, Plaque Assay, Immunofluorescence, Imaging, Standard Deviation

    3) Product Images from "Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning"

    Article Title: Selection and Characterization of Monoclonal Antibodies Targeting Middle East Respiratory Syndrome Coronavirus through a Human Synthetic Fab Phage Display Library Panning

    Journal: Antibodies

    doi: 10.3390/antib8030042

    Characterization of anti-MERS-S2P IgGs. ( A ) Immunofluorescence assay of anti-MERS-S2P IgGs on cell lines infected with various human coronaviruses (hCoVs) to determine their MERS-CoV specificity. Scale bar, 200 μm. ( B ) Size-exclusion chromatography analysis of S2A3 (IgG) and S2D5 (IgG). The molecular weights (kDa) of the molecular mass markers are shown above the corresponding peaks at the top chromatogram. ( C ) Surface plasmon resonance (SPR) analysis of S2A3 (IgG) and S2D5 (IgG) on a MERS-S2P-immobilized sensor chip to determine their apparent binding affinities. The fitted-lines are marked by red.
    Figure Legend Snippet: Characterization of anti-MERS-S2P IgGs. ( A ) Immunofluorescence assay of anti-MERS-S2P IgGs on cell lines infected with various human coronaviruses (hCoVs) to determine their MERS-CoV specificity. Scale bar, 200 μm. ( B ) Size-exclusion chromatography analysis of S2A3 (IgG) and S2D5 (IgG). The molecular weights (kDa) of the molecular mass markers are shown above the corresponding peaks at the top chromatogram. ( C ) Surface plasmon resonance (SPR) analysis of S2A3 (IgG) and S2D5 (IgG) on a MERS-S2P-immobilized sensor chip to determine their apparent binding affinities. The fitted-lines are marked by red.

    Techniques Used: Immunofluorescence, Infection, Size-exclusion Chromatography, SPR Assay, Chromatin Immunoprecipitation, Binding Assay

    Output of the panning of the phage-displayed synthetic Fab library on MERS-S2P. ( A ) Monitoring of phage titers over three rounds (R1–R3) of panning. Black and gray bars indicate a ratio of phage output to input titers presented as a percentage (%) from panning on MERS-S2P-immobilized and -non-immobilized surfaces, respectively. The ratio of output to input (%) = (phage output titer ÷ phage input titer) × 100. ( B ) Phage ELISAs performed on MERS-S2P-, SARS-CoV spike protein-, a CoV spike protein-immobilized surfaces (blue, red, and green, respectively). ( C ) Amino acid sequences of three unique clones identified from panning (left) and their relative frequencies (%) (right). The sequences were aligned using the Kabat numbering system [ 38 ]. ELISA, enzyme-linked immunosorbent assay; MERS-S2P, Middle East respiratory syndrome-CoV S2 subunit protein; SARS-SP, severe acute respiratory syndrome-CoV S protein; HKU1-SP, hCoV HKU1 S protein; CoV, coronavirus; CDR, complementarity-determining region; FR, framework region.
    Figure Legend Snippet: Output of the panning of the phage-displayed synthetic Fab library on MERS-S2P. ( A ) Monitoring of phage titers over three rounds (R1–R3) of panning. Black and gray bars indicate a ratio of phage output to input titers presented as a percentage (%) from panning on MERS-S2P-immobilized and -non-immobilized surfaces, respectively. The ratio of output to input (%) = (phage output titer ÷ phage input titer) × 100. ( B ) Phage ELISAs performed on MERS-S2P-, SARS-CoV spike protein-, a CoV spike protein-immobilized surfaces (blue, red, and green, respectively). ( C ) Amino acid sequences of three unique clones identified from panning (left) and their relative frequencies (%) (right). The sequences were aligned using the Kabat numbering system [ 38 ]. ELISA, enzyme-linked immunosorbent assay; MERS-S2P, Middle East respiratory syndrome-CoV S2 subunit protein; SARS-SP, severe acute respiratory syndrome-CoV S protein; HKU1-SP, hCoV HKU1 S protein; CoV, coronavirus; CDR, complementarity-determining region; FR, framework region.

    Techniques Used: Clone Assay, Enzyme-linked Immunosorbent Assay

    Detection of MERS-S2P using S2A3 (IgG) on ACCEL ELISA™ plates. ( A ) Schematic depicting the sandwich ELISA format to detect MERS-S2P using S2A3 (IgG) (capture antibody) and rabbit anti-MERS-CoV IgG (detection antibody) on ACCEL ELISA™ plates. ( B ) ELISA detection of MERS-S2P on a capture antibody (S2A3 (IgG)) immobilized using three different concentrations (3 µg/mL, 5 µg/mL, and 10 µg/mL) on ACCEL ELISA™ plates. The goodness of fit is indicated by the R 2 value. LOD, limit of detection.
    Figure Legend Snippet: Detection of MERS-S2P using S2A3 (IgG) on ACCEL ELISA™ plates. ( A ) Schematic depicting the sandwich ELISA format to detect MERS-S2P using S2A3 (IgG) (capture antibody) and rabbit anti-MERS-CoV IgG (detection antibody) on ACCEL ELISA™ plates. ( B ) ELISA detection of MERS-S2P on a capture antibody (S2A3 (IgG)) immobilized using three different concentrations (3 µg/mL, 5 µg/mL, and 10 µg/mL) on ACCEL ELISA™ plates. The goodness of fit is indicated by the R 2 value. LOD, limit of detection.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

    Related Articles

    Incubation:

    Article Title: Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusion-stabilized spike protein of Middle East respiratory syndrome coronavirus
    Article Snippet: .. As positive controls, a rabbit polyclonal anti-MERS-CoV spike protein antibody (Sino biological Inc., Beijing, China) for MERS-CoV, a mouse anti-229E coronavirus nucleoprotein OC-43 antibody (MERCK, Darmstadt, Germany) for HCoV-229E, a mouse anti-coronavirus antibody, hCoV OC-43 (LifeSpan BioScience, Seattle, WA, USA) for HCoV-OC43, a rabbit polyclonal anti-hCoV-HKU1 spike protein antibody (Sino Biological Inc., Beijing, China) for hCoV-NL63, a human serum of a Korean SARS-CoV-2 convalescent person were also incubated. ..

    Article Title: Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2
    Article Snippet: .. The membrane was sealed in phosphate-buffered saline with Tween-20 (PBST) with 5% skim milk at 37°C for 2 h and subsequently incubated overnight with the primary antibodies anti-N protein rabbit monoclonal Ab (Sino Biological) (1:1000 dilution) and anti-S protein rabbit polyclonal Ab (Sino Biological) (1:1000 dilution) and human convalescent sera from patients (1:500 dilution) at 4°C. ..

    Article Title: Bromodomain and extraterminal protein inhibitor, apabetalone (RVX-208), reduces ACE2 expression and attenuates SARS-CoV-2 infection in vitro
    Article Snippet: .. The cells were incubated with anti-Spike protein Rab (Sino Biological) at 1:1000 in blocking solution overnight at 4°C, followed by incubation with 1:2000 diluted Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher) for 1 h at room temperature. ..

    Article Title: Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion
    Article Snippet: .. Generation of stable cell lines with hACE-2-mCherry, SARS-CoV-2 Spike-full and -C terminal Δ19-EGFP 293T cells were transfected with Invitrogen™ ViraPower™ Lentiviral Packaging Mix as followed: 2 × 106 293T cells were seeded the day before and mixed with 1 ml Optimal MEM transfection solution with 45 μl Genejuice (Millipore) containing 3.75 μg pCMV-dR8.91, 2.5 μg pMD2.G-VSVG, and either 4.17 μg CHC16-pHR_hACE2_mCherry, CHC17-pHR_SARS-CoV-2 Swt_EGFP, or CHC-18-pHR_SARS-CoV-2 S-Δ19_EGFP at RT for 15 min, and then incubated with fresh D10 medium (10% FBS in DMEM without antibiotics) at 37℃ and 5% (v/v) CO2 for 12 h. Transfected 293T cell media was changed after 24 h and incubated for another 48–72 h. The lentivirus supernatant was harvested, filtered (0.45 μm filter Millipore) and transduced into 293T, A549, HepG2, and SK-Hep1 cells with serum-free DMEM for 12 h. The transduced cell media was changed with fresh complete antibiotic-containing D10 medium for another 48–72 h. Transduced cells were flow-sorted by EGFP/mCherry expression, or protein expression determined by anti-Spike protein RBD domain antibody (rabbit IgG, Sino Biological, 40021-T62) or anti-hACE2 antibody (goat IgG, R & D Systems, AF933) followed by fluorophore-conjugated goat anti-rabbit (Invitrogen, 111-585-144) or donkey anti-goat secondary antibody (Jackson Immuno Research, 705-545-003). ..

    Immunolabeling:

    Article Title: Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential
    Article Snippet: .. At 20 h posttransfection, the cells were fixed with 4% PFA, permeabilized with 0.3% Triton X-100, and immunolabeled using primary polyclonal antibody against MERS-CoV spike protein (SinoBiological) and secondary Alexa Fluor 488-conjugated goat anti-rabbit antibody (Thermo Fisher). ..

    Blocking Assay:

    Article Title: Bromodomain and extraterminal protein inhibitor, apabetalone (RVX-208), reduces ACE2 expression and attenuates SARS-CoV-2 infection in vitro
    Article Snippet: .. The cells were incubated with anti-Spike protein Rab (Sino Biological) at 1:1000 in blocking solution overnight at 4°C, followed by incubation with 1:2000 diluted Alexa Fluor 488 conjugated secondary antibody (Thermo Fisher) for 1 h at room temperature. ..

    Recombinant:

    Article Title: Development of a SERS-based lateral flow immunoassay for rapid and ultra-sensitive detection of anti-SARS-CoV-2 IgM/IgG in clinical samples
    Article Snippet: .. SARS-CoV-2 S protein (recombinant) and S protein antibody (Ab) were obtained from Sino Biological Inc. (Beijing, China). ..

    Stable Transfection:

    Article Title: Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion
    Article Snippet: .. Generation of stable cell lines with hACE-2-mCherry, SARS-CoV-2 Spike-full and -C terminal Δ19-EGFP 293T cells were transfected with Invitrogen™ ViraPower™ Lentiviral Packaging Mix as followed: 2 × 106 293T cells were seeded the day before and mixed with 1 ml Optimal MEM transfection solution with 45 μl Genejuice (Millipore) containing 3.75 μg pCMV-dR8.91, 2.5 μg pMD2.G-VSVG, and either 4.17 μg CHC16-pHR_hACE2_mCherry, CHC17-pHR_SARS-CoV-2 Swt_EGFP, or CHC-18-pHR_SARS-CoV-2 S-Δ19_EGFP at RT for 15 min, and then incubated with fresh D10 medium (10% FBS in DMEM without antibiotics) at 37℃ and 5% (v/v) CO2 for 12 h. Transfected 293T cell media was changed after 24 h and incubated for another 48–72 h. The lentivirus supernatant was harvested, filtered (0.45 μm filter Millipore) and transduced into 293T, A549, HepG2, and SK-Hep1 cells with serum-free DMEM for 12 h. The transduced cell media was changed with fresh complete antibiotic-containing D10 medium for another 48–72 h. Transduced cells were flow-sorted by EGFP/mCherry expression, or protein expression determined by anti-Spike protein RBD domain antibody (rabbit IgG, Sino Biological, 40021-T62) or anti-hACE2 antibody (goat IgG, R & D Systems, AF933) followed by fluorophore-conjugated goat anti-rabbit (Invitrogen, 111-585-144) or donkey anti-goat secondary antibody (Jackson Immuno Research, 705-545-003). ..

    Transfection:

    Article Title: Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion
    Article Snippet: .. Generation of stable cell lines with hACE-2-mCherry, SARS-CoV-2 Spike-full and -C terminal Δ19-EGFP 293T cells were transfected with Invitrogen™ ViraPower™ Lentiviral Packaging Mix as followed: 2 × 106 293T cells were seeded the day before and mixed with 1 ml Optimal MEM transfection solution with 45 μl Genejuice (Millipore) containing 3.75 μg pCMV-dR8.91, 2.5 μg pMD2.G-VSVG, and either 4.17 μg CHC16-pHR_hACE2_mCherry, CHC17-pHR_SARS-CoV-2 Swt_EGFP, or CHC-18-pHR_SARS-CoV-2 S-Δ19_EGFP at RT for 15 min, and then incubated with fresh D10 medium (10% FBS in DMEM without antibiotics) at 37℃ and 5% (v/v) CO2 for 12 h. Transfected 293T cell media was changed after 24 h and incubated for another 48–72 h. The lentivirus supernatant was harvested, filtered (0.45 μm filter Millipore) and transduced into 293T, A549, HepG2, and SK-Hep1 cells with serum-free DMEM for 12 h. The transduced cell media was changed with fresh complete antibiotic-containing D10 medium for another 48–72 h. Transduced cells were flow-sorted by EGFP/mCherry expression, or protein expression determined by anti-Spike protein RBD domain antibody (rabbit IgG, Sino Biological, 40021-T62) or anti-hACE2 antibody (goat IgG, R & D Systems, AF933) followed by fluorophore-conjugated goat anti-rabbit (Invitrogen, 111-585-144) or donkey anti-goat secondary antibody (Jackson Immuno Research, 705-545-003). ..

    Expressing:

    Article Title: Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion
    Article Snippet: .. Generation of stable cell lines with hACE-2-mCherry, SARS-CoV-2 Spike-full and -C terminal Δ19-EGFP 293T cells were transfected with Invitrogen™ ViraPower™ Lentiviral Packaging Mix as followed: 2 × 106 293T cells were seeded the day before and mixed with 1 ml Optimal MEM transfection solution with 45 μl Genejuice (Millipore) containing 3.75 μg pCMV-dR8.91, 2.5 μg pMD2.G-VSVG, and either 4.17 μg CHC16-pHR_hACE2_mCherry, CHC17-pHR_SARS-CoV-2 Swt_EGFP, or CHC-18-pHR_SARS-CoV-2 S-Δ19_EGFP at RT for 15 min, and then incubated with fresh D10 medium (10% FBS in DMEM without antibiotics) at 37℃ and 5% (v/v) CO2 for 12 h. Transfected 293T cell media was changed after 24 h and incubated for another 48–72 h. The lentivirus supernatant was harvested, filtered (0.45 μm filter Millipore) and transduced into 293T, A549, HepG2, and SK-Hep1 cells with serum-free DMEM for 12 h. The transduced cell media was changed with fresh complete antibiotic-containing D10 medium for another 48–72 h. Transduced cells were flow-sorted by EGFP/mCherry expression, or protein expression determined by anti-Spike protein RBD domain antibody (rabbit IgG, Sino Biological, 40021-T62) or anti-hACE2 antibody (goat IgG, R & D Systems, AF933) followed by fluorophore-conjugated goat anti-rabbit (Invitrogen, 111-585-144) or donkey anti-goat secondary antibody (Jackson Immuno Research, 705-545-003). ..

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    Sino Biological monoclonal rabbit anti mers cov spike protein s1 antibody
    Identification of an H2-d restricted T cell epitope in <t>MERS-CoV</t> N protein; ( a–d ) Groups of BALB/c mice ( n = 3 to 8) were vaccinated in a prime-boost regime with 10 8 PFU of MVA-MERS-N via i.p. ( a ) or i.m. ( b–d ) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. ( a-b ) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. ( c–d ) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide ( c ) or F2L 26-34 peptide ( d ) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *
    Monoclonal Rabbit Anti Mers Cov Spike Protein S1 Antibody, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal rabbit anti mers cov spike protein s1 antibody/product/Sino Biological
    Average 86 stars, based on 1 article reviews
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    94
    Sino Biological mers cov nucleocapsid protein antibody rabbit pab
    IHC detection of virus antigen expression in mouse tissue after challenge with <t>MERS-CoV.</t> Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.
    Mers Cov Nucleocapsid Protein Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov nucleocapsid protein antibody rabbit pab/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov nucleocapsid protein antibody rabbit pab - by Bioz Stars, 2021-09
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    94
    Sino Biological mers cov spike antibody rabbit pab
    Pseudo-particle assays of <t>MERS-CoV</t> S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.
    Mers Cov Spike Antibody Rabbit Pab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov spike antibody rabbit pab/product/Sino Biological
    Average 94 stars, based on 1 article reviews
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    94
    Sino Biological mers cov spike antibody rabbit mab
    <t>MERS-CoV</t> spike-pseudotyped neutralisation p values were calculated using Kruskall-Wallis with Dunn's multiple comparison post test. The dashed lines represent lower limit of detection under our experimental condition. Data points represent geometric means, and error bars represent 95% CIs. MERS-CoV=Middle East respiratory syndrome coronavirus.
    Mers Cov Spike Antibody Rabbit Mab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mers cov spike antibody rabbit mab/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mers cov spike antibody rabbit mab - by Bioz Stars, 2021-09
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    Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; ( a–d ) Groups of BALB/c mice ( n = 3 to 8) were vaccinated in a prime-boost regime with 10 8 PFU of MVA-MERS-N via i.p. ( a ) or i.m. ( b–d ) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. ( a-b ) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. ( c–d ) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide ( c ) or F2L 26-34 peptide ( d ) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Identification of an H2-d restricted T cell epitope in MERS-CoV N protein; ( a–d ) Groups of BALB/c mice ( n = 3 to 8) were vaccinated in a prime-boost regime with 10 8 PFU of MVA-MERS-N via i.p. ( a ) or i.m. ( b–d ) application. Mice immunized with non-recombinant MVA (MVA) and PBS served as negative controls. ( a-b ) Splenocytes were stimulated with individual 8-11-mer peptides and IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. ( c–d ) Splenocytes were stimulated with positive MERS-CoV N 10.2 peptide ( c ) or F2L 26-34 peptide ( d ) and IFN-γ producing CD8+ or CD4+ T cells were measured using intracellular cytokine staining assay and FACS analysis. The lines represent means. *

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Mouse Assay, Recombinant, Enzyme-linked Immunospot, Staining, FACS

    Analysis of recombinant MVA-MERS proteins; ( a ) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; ( b – d ) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody ( b ) or polyclonal sera from MERS-CoV infected rabbits ( c ) or cynomolgus macaques ( d ). Arrows indicate the N- or S-specific protein bands.

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Analysis of recombinant MVA-MERS proteins; ( a ) Western Blot analysis of MERS-CoV N protein produced in CEF or HaCat cells. Lysates from cells infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at eight, 12, or 24 hpi. Proteins were analyzed by immunoblotting with a monoclonal anti-MERS-N antibody; ( b – d ) Western Blot analysis of MERS-CoV N and S proteins produced in CEF. Total cell extracts from CEF infected with recombinant MVA (MVA-MERS-N, MVA-MERS-S) or non-recombinant MVA (MVA) at a MOI of five, or from non-infected cells (mock) were prepared at 24 hpi. Cell lysates and proteins were tested by immunoblotting using monoclonal anti MERS-N and anti MERS-S antibody ( b ) or polyclonal sera from MERS-CoV infected rabbits ( c ) or cynomolgus macaques ( d ). Arrows indicate the N- or S-specific protein bands.

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Recombinant, Western Blot, Produced, Infection

    Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; ( a – b ) groups of BALB/c mice ( n = 2 to 6) were vaccinated twice (21-day interval) by i.p. ( a ) or i.m. ( b ) application with 10 8 plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.

    Journal: Viruses

    Article Title: CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice

    doi: 10.3390/v10120718

    Figure Lengend Snippet: Screening for H2-d restricted T cell epitopes in MERS-CoV N protein using matrix peptide pools; ( a – b ) groups of BALB/c mice ( n = 2 to 6) were vaccinated twice (21-day interval) by i.p. ( a ) or i.m. ( b ) application with 10 8 plaque-forming-units (PFU) of recombinant MVA-MERS-N (MVA-N). Mice inoculated with non-recombinant MVA (MVA) or phosphate-buffered saline (PBS) were used as controls. Splenocytes were restimulated in vitro with pools of overlapping peptides corresponding to MERS-CoV N protein. IFN-γ spot-forming CD8+ T cells (IFN-γ SFC) were measured by ELISPOT. The lines represent means.

    Article Snippet: After 1 h blocking in a phosphate buffered saline (PBS) buffer containing 1% (w/v) non-fat dried milk and 0.1% (v/v) NP-40 detergent, the blots were incubated with monoclonal mouse anti-MERS-CoV Nucleocapsid antibody (Sino Biological, Beijing, China, 1:1000), monoclonal rabbit anti-MERS-CoV Spike Protein S1 Antibody (Sino Biological, 1:500), or polyclonal sera from MERS-CoV infected rabbits or cynomolgus macaques (kindly provided by Dr. Bart Haagmans, Erasmus Medical Center, Rotterdam, 1:1000) [ ] as primary antibodies.

    Techniques: Mouse Assay, Recombinant, In Vitro, Enzyme-linked Immunospot

    IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Immunohistochemistry, Expressing

    rNTD or rRBD vaccination reduced respiratory tract pathology in mice after MERS-CoV challenge. Representative results of hematoxylin-eosin (HE) staining in the lung (A – C) and trachea (D – F) of mock-treated or immunized mice. Severe lesions including the loss of pulmonary alveolus (represented by the white vacuole) and diffuse inflammatory cell infiltration (represented by the dark purple point) are shown (figure A). In contrast, milder lesions were observed among mice immunized with rRBD (figure B) or NTD (figure C), as the pulmonary alveolus was highly visible with less inflammatory cell infiltration. Inflammatory cell infiltration and impaired epithelium of the tunica mucosa bronchiorum were seen in the mock group (D). rRBD (E) or rNTD (F) alleviated the pathologic damage in the trachea of immunized mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: rNTD or rRBD vaccination reduced respiratory tract pathology in mice after MERS-CoV challenge. Representative results of hematoxylin-eosin (HE) staining in the lung (A – C) and trachea (D – F) of mock-treated or immunized mice. Severe lesions including the loss of pulmonary alveolus (represented by the white vacuole) and diffuse inflammatory cell infiltration (represented by the dark purple point) are shown (figure A). In contrast, milder lesions were observed among mice immunized with rRBD (figure B) or NTD (figure C), as the pulmonary alveolus was highly visible with less inflammatory cell infiltration. Inflammatory cell infiltration and impaired epithelium of the tunica mucosa bronchiorum were seen in the mock group (D). rRBD (E) or rNTD (F) alleviated the pathologic damage in the trachea of immunized mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Mouse Assay, Staining

    Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Vaccine

    Article Title: The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

    doi: 10.1016/j.vaccine.2016.11.064

    Figure Lengend Snippet: Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Rabbit-serum-derived polyclonal antibody against nucleoprotein (cat: 100213-RP02; Sino Biological Inc., Beijing, CHN) was incubated with the sections at 1:1000 dilution; goat anti-rabbit (cat: pv-9001; ZSGB-Bio, Beijing, CHN) secondary antibody was used at 1:2000, and sections were evaluated using light microscopy.

    Techniques: Recombinant, Expressing, Purification, SDS Page, Western Blot, Staining, Labeling, Molecular Weight, Marker, Mouse Assay, Enzyme-linked Immunospot, Crocin Bleaching Assay

    Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Pseudo-particle assays of MERS-CoV S WT and mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective S mutants. After 72 h, infected cells were lysed and assessed for luciferase activity. (A) PP infectivity of Huh 7 cells. (B) Infectivity of PP carrying the D922A S protein. Δenv and VSV-G served as representative controls for all PP assays (C) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium containing either 50 µM calcium chelator BAPTA-AM ordimethyl sulfoxide (DMSO) for 1 h. Cells were then infected with their respective PP in the presence of BAPTA-AM or DMSO for 2 h and grown for 72 h before assessing for luciferase activity. (D) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells were pre-treated with growth medium either with or without 1.8 mM Ca 2+ for 1 h. Infection protocol is as described above except PP were treated with 1.5 mM EGTA for calcium chelation. Infectivity was normalized such that WT PP infectivity is 1. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test, as indicated. * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Infection, Luciferase, Activity Assay, Standard Deviation

    ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: ESR and ITC analysis of the MERS-CoV FP. A-B. Plots of order parameters of DPPTC (A), and 5PC (B) versus peptide:lipid ratio (P/L ratio) of MERS FP or SARS FP in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP, 1 mM Ca 2+ and at pH 5; red, MERS FP calcium-less buffer with 1 mM EGTA and at pH 5; blue, SARS FP, 1 mM Ca 2+ at pH 5, and purple, scrambled peptide, 1 mM Ca 2+ and at pH 5. (C) Plot of difference of order parameters of DPPTC with and without 1% peptide binding (ΔS0) versus Ca 2+ concentration in POPC/POPS/Chol=3/1/1 MLVs in buffer with 150 mM NaCl at 25°C. Black, MERS FP; blue, SARS FP; and green, scrambled peptide. The experiments were typically repeated two to three times. The typical uncertainties found for S 0 ranges from 1-5 × 10 −3 , while the uncertainties from repeated experiments were 5-8 × 10 −3 or less than ±0.01. We show the standard deviation bars in Panel A and B. (D) ITC analysis of Ca 2+ binding to MERS-CoV FP. The peptides were titrated with CaCl 2 . The integrated data represent the enthalpy change per mole of injectant, ΔH, in units of kJ/mol as a function of the molar ratio. Data points and fitted data are overlaid. The fitting is based on the one-site model.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Binding Assay, Concentration Assay, Standard Deviation

    Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Sequence and model of MERS-CoV S fusion loop. (A) Sequences of SARS-CoV S Urbani and MERS-CoV S EMC/2012 fusion peptides (FP). FP1 and FP2 designate the two different domains in the FP as described previously (Lai Millet). Sequences below illustrate the mutations that were introduced in the MERS-CoV S protein via site-directed mutagenesis. In red are the negatively charged residues D and E, in green are the A substitutions. (B) Modeling of the MERS-CoV S monomer with an emphasis on the FP. Negatively charges Ds and E are depicted as atomic bonds in red. The S2’ site is orange and the FP1 and FP2 domains are labeled blue and pink, respectively.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Sequencing, Mutagenesis, Labeling

    Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Western blot analysis of S proteins incorporated into PPs. 1 ml of DMEM containing PPs per each tested S protein were ultra-centrifuged, washed in PBS and resuspended in SDS Laemmli Buffer. Incorporated S proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Western Blot, SDS Page

    Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Pseudo-particle assays of MERS-CoV S WT and E891A/D896A, E891A/D902A and E891A/D896A/D902A mutants. Huh-7 cells were infected with MLV-based pseudo-particles (PP) carrying MERS-CoV S WT or one of the respective mutants. Infectivity was normalized to WT sample. Error bars represent standard deviation (n = 3). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against the respective mutant (for B and C the untreated WT was compared to each sample). * = p > 0.5, ** = p > 0.05, *** = p > 0.005. (A) Infectivity of PPs without pre-treatment of cells. (B) Impact of intracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 C . (C) Impact of extracellular Ca 2+ on MERS-CoV fusion. Cells and PPs were treated as described for Figure 5 D .

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Infection, Standard Deviation, Mutagenesis

    Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Immunofluorescence assay of MERS-CoV S WT and mutants. (A) Vero cells were transfected with plasmid DNA encoding for the respective MERS-CoV S variants and the DPP4 binding receptor and grown for 18 h. As Vero cells express endogenous proteases, which cleaves MERS-CoV S for fusion, no further protease treatment was needed to induce syncytia formation. WT + protease inhibitor indicates the condition in which protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection to block fusion. Syncytia was visualized using immunofluorescence microscopy by staining the MERS-CoV S with a polyclonal anti-S antibody (in green) and the nuclei with 4′,6-diamidino-2-phenylindole (DAPI, in blue). Images were taken at a magnification of 25x. (B) Quantification of syncytia. Nuclei of 9 syncytia were counted and the average number of nuclei per syncytia was calculated. Error bars represent standard deviation (n = 9). Statistical analysis was performed using an unpaired student’s t-test comparing the WT against each of the respective mutant * = p > 0.5, ** = p > 0.05, *** = p > 0.005.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Immunofluorescence, Transfection, Plasmid Preparation, Binding Assay, Protease Inhibitor, Concentration Assay, Blocking Assay, Microscopy, Staining, Standard Deviation, Mutagenesis

    Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.

    Journal: bioRxiv

    Article Title: Ca2+ ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity

    doi: 10.1101/2019.12.18.881391

    Figure Lengend Snippet: Protein expression and trypsin-mediated cleavage of MERS-CoV S WT and mutants. (A) Plasmid DNA encoding MERS-CoV S WT EMC/2012 was transfected in HEK293T cells. The protease inhibitor dec-RVKR-CMK at a concentration of 75 µM was added at the time of transfection, as indicated. After 18 h, transfected cells were treated with 0.8 nM TPCK-treated trypsin, as indicated. Proteins were subsequently isolated via cell-surface biotinylation. The cell surface proteins were analyzed using SDS-PAGE and detected using a Western blot with MERS-CoV S antibodies. (B) and (C) MERS-CoV S mutant proteins with indicated A substitutions were expressed in HEK293T cells. Protease inhibitor dec-RVKR-CMK was added at the time of transfection and after 18 h, cells were treated with TPCK-treated trypsin, as indicated. Cell surface proteins were isolated and analyzed as described above. Full length S proteins are visible at approx. 250 kDa. S1/S2 cleaved S protein are visible at approx. 115 kDa.

    Article Snippet: S protein was detected using the MERS-CoV S rabbit polyclonal antibody (Sino Biological, Cat No: 40069-RP01) as the primary antibody, and AlexaFluor 488-labeled anti-rabbit secondary antibody (Invitrogen).

    Techniques: Expressing, Plasmid Preparation, Transfection, Protease Inhibitor, Concentration Assay, Isolation, SDS Page, Western Blot, Mutagenesis

    MERS-CoV spike-pseudotyped neutralisation p values were calculated using Kruskall-Wallis with Dunn's multiple comparison post test. The dashed lines represent lower limit of detection under our experimental condition. Data points represent geometric means, and error bars represent 95% CIs. MERS-CoV=Middle East respiratory syndrome coronavirus.

    Journal: The Lancet. Infectious Diseases

    Article Title: Safety and immunogenicity of a candidate Middle East respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial

    doi: 10.1016/S1473-3099(20)30160-2

    Figure Lengend Snippet: MERS-CoV spike-pseudotyped neutralisation p values were calculated using Kruskall-Wallis with Dunn's multiple comparison post test. The dashed lines represent lower limit of detection under our experimental condition. Data points represent geometric means, and error bars represent 95% CIs. MERS-CoV=Middle East respiratory syndrome coronavirus.

    Article Snippet: A commercially available neutralising monoclonal antibody (40069-R723, Sino Biological, Beijing, China) was used as a positive control.

    Techniques: