All SARS-CoV-2 infection experiments were performed in a biosafety level-3 laboratory. Vero E6 cells were seeded onto 96-well plates overnight and grown into confluent monolayers. Fifty microliters of 10-fold-diluted SARS-CoV-2 stock or supernatant of lung homogenate was added into 96-well plate and adsorbed at 37 °C for 1 h with rocking every 10 min. Then the virus or supernatant of lung homogenate were removed and covered with 100 μL Minimum Essential Medium (MEM) containing 1.2% Carboxymethylcellulose (1.2% CMC). After 24 h post infection, the overlay was discarded and the cell monolayer was fixed with 4% paraformaldehyde solution for 2 h at room temperature (RT). After permeabilized with 0.2% Triton X-100 for 20 min at RT, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological Inc) as the primary antibody and HRP-conjugated goat anti-rabbit IgG(H + L) (Jackson ImmunoResearch) as the secondary antibody in 37 °C for 1 h, respectively. The reactions were developed with KPL TrueBlue Peroxidase substrates. The numbers of SARS-CoV-2 foci were calculated using CTL ImmunoSpot S6 Ultra reader (Cellular Technology Ltd).
Cross reactive rabbit anti sars cov n igg
Manufactured by Sino Biological
The Cross-reactive rabbit anti-SARS-CoV-N IgG is a laboratory reagent that can be used for the detection and identification of SARS-CoV-N protein. It is produced by immunizing rabbits with the SARS-CoV-N protein and purifying the resulting IgG antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ctl immunospot s6 ultra reader, by Cellular Technology (3 mentions)
Hrp conjugated goat anti rabbit igg h l antibody, by Jackson ImmunoResearch (2 mentions)
Elispot reader, by Cellular Technology (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg h l, by Jackson ImmunoResearch (2 mentions)
Hrp conjugated goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Vero e6 cell, by American Type Culture Collection (1 mentions)
5 protocols using cross reactive rabbit anti sars cov n igg
1
SARS-CoV-2 Focus Forming Assay
2
SARS-CoV-2 Neutralization Assay Protocol
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SARS-CoV-2 focus reduction neutralization test was performed in a certified Biosafety Level 3 laboratory, as previously described with the following modifications (Ju et al., 2020 (link)). Briefly, a clinical isolate (Beta/Shenzhen/SZTH-003/2020, EPI_ISL_406594 at GISAID) previously obtained from a nasopharyngeal swab of an infected patient was used for the analysis. Serial concentrations of Nbs were mixed with 75 μL of SARS-CoV-2 (8 × 103 focus forming unit/ml, FFU/ml) in 96-well microwell plates and incubated at 37°C for 1 h. The mixtures were then transferred to 96-well plates seeded with Vero E6 cells and incubated at 37°C for 1 h. Next, the inoculums were removed prior to the addition of the overlay media (100 μL MEM containing 1.6% carboxymethylcellulose, CMC) and the plates were then incubated at 37°C for 24 h. Cells were fixed with 4% paraformaldehyde solution for 30 min, and then the overlays were removed. Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc) for 1 h at room temperature before the addition of HRP-conjugated goat anti-rabbit IgG (H+L) antibody (Jackson ImmunoResearch) and further incubated at room temperature. The foci were stained with KPL TrueBlue Peroxidase substrates (SeraCare Life Sciences Inc.) and were counted with an EliSpot reader (Cellular Technology Ltd.).
3
SARS-CoV-2 Focus-Forming Assay Protocol
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For the focus-forming assay (FFA), all SARS-CoV-2 live virus infection experiments were performed in a biosafety level 3 (BSL3) laboratory. Vero E6 cells were seeded onto 96-well plates overnight and grown into confluent monolayers. Fifty microliters of 10-fold-diluted SARS-CoV-2 stock or supernatant of lung homogenate was added into a 96-well plate and adsorbed at 37°C for 1 h with agitation every 10 min. Then, the virus or supernatant of lung homogenate was removed and covered with 100 μL minimum essential medium (MEM) containing 1.2% carboxymethylcellulose (CMC). Twenty-four hours postinfection, the overlay was discarded and the cell monolayer was fixed with 4% paraformaldehyde solution for 2 h at room temperature. After being permeabilized with 0.2% Triton X-100 for 20 min at room temperature, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc.) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) as the secondary antibody at 37°C for 1 h. The reactions were developed with KPL TrueBlue peroxidase substrates. The numbers of SARS-CoV-2 foci were calculated using CTL ImmunoSpot S6 Ultra reader (Cellular Technology, Ltd.), and titers of the virus were expressed as focus-forming units (FFU) per milliliter.
4
SARS-CoV-2 Neutralization Assay Protocol
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SARS-CoV-2 FRNT was performed in a certified biosafety level 3 lab as previously described.13 (link) Fifty-μl serum samples were serially (1:4) diluted, mixed with 50 μl of SARS-CoV-2 (100 focus-forming unit, FFU) in 96-well microwell plates, and incubated for 1 h at 37 °C. Mixtures were then transferred to 96-well plates seeded with Vero E6 cells (ATCC, Manassas, VA) for 1 h at 37 °C. Inoculums were then removed before adding the overlay media (100 μl MEM containing 1.2% carboxymethylcellulose, CMC). The plates were then incubated at 37 °C for 24 h. Overlays were removed and cells were fixed with 4% paraformaldehyde solution for 30 min. Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-N IgG (Cat: 40143-R001, Sino Biological, Inc, Beijing) for 1 h at room temperature before adding HRP-conjugated goat anti-rabbit IgG(H + L) antibody (1:4000 dilution) (Code: 111-035-144, Jackson ImmunoResearch, West Grove, PA). Cells were further incubated at room temperature. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences Inc., Milford, MA). The numbers of SARS-CoV-2 foci were calculated using an EliSpot reader (Cellular Technology Ltd., Shaker Heights, OH). IC50 of virus neutralization was calculated using the 4-parameter logistic model.
5
SARS-CoV-2 Focus-Forming Assay Protocol
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For the focus-forming assay (FFA), all SARS-CoV-2 live virus infection experiments were performed in a biosafety level 3 (BSL3) laboratory. Vero E6 cells were seeded onto 96-well plates overnight and grown into confluent monolayers. Fifty microliters of 10-fold-diluted SARS-CoV-2 stock or supernatant of lung homogenate was added into a 96-well plate and adsorbed at 37°C for 1 h with agitation every 10 min. Then, the virus or supernatant of lung homogenate was removed and covered with 100 μL minimum essential medium (MEM) containing 1.2% carboxymethylcellulose (CMC). Twenty-four hours postinfection, the overlay was discarded and the cell monolayer was fixed with 4% paraformaldehyde solution for 2 h at room temperature. After being permeabilized with 0.2% Triton X-100 for 20 min at room temperature, the plates were sequentially stained with cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological, Inc.) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch) as the secondary antibody at 37°C for 1 h. The reactions were developed with KPL TrueBlue peroxidase substrates. The numbers of SARS-CoV-2 foci were calculated using CTL ImmunoSpot S6 Ultra reader (Cellular Technology, Ltd.), and titers of the virus were expressed as focus-forming units (FFU) per milliliter.
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