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Detection of SARS-CoV-2 Variant Proteins with Spike RBD Antibodies by Direct ELISA. Coating Antigen: SARS-CoV-2 full length spike proteins, including WT, UK variant (B.1.1.7), SA variant (B.1.135) and Brazil (P.1). Dilution: 10-3000 ng/mL. Incubate at 4 °C overnight. Detection Antibodies: SARS-CoV-2 Spike RBD Antibody, orb1239994, 2 µg/mL, incubate at RT for 1 hr. Secondary Antibodies: Goat anti-rabbit HRP at 1:20000, incubate at RT for 1 hr. Immunogen region of antibody (orb1239994) includes site 501N that was mutated in all three variants. orb1239994 has low binding affinity for all three variants as compared to WT. |
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ELISA Validation with SARS |
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Immunohistochemistry Validation of SARS-CoV-2 (COVID-19) Spike RBD in COVID-19 Patient Lung. Immunohistochemical analysis of paraffin-embedded COVID-19 patient lung tissue using anti-SARS-CoV-2 (COVID-19) Spike RBD antibody (orb1239994, 0.5 µg/mL). Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT, antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4 °C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin. Strong signal of SARS-COV-2 Spike RBD protein was observed in macrophage of COVID-19 patient lung, but not in non-COVID-19 patient lung. |
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Western Blot Validation with SARS-CoV-2 (COVID-19) Spike RBD Recombinant Protein. Loading: 30 ng per lane of SARS-CoV-2 (COVID-19) Spike RBD recombinant protein, orb1231588. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD, orb1239994, 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: 1 µg/mL and Lane 2: 2 µg/mL. |
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ELISA Validation with SARS-CoV-2 (COVID-19) Spike RBD Recombinant Protein. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD antibody, orb1239994 (1 µg/mL). A direct ELISA was performed using SARS-CoV-2 (COVID-19) Spike RBD recombinant protein (orb1231588) as coating antigen and the anti-SARS-CoV-2 (COVID-19) Spike RBD antibody as the capture. |
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Immunofluorescence Validation of SARS-CoV-2 (COVID-19) Spike RBD in 293 Transfected Cells. Immunofluorescent analysis of 4% paraformaldehyde-fixed Spike transfected 293 cells labeling SARS-CoV-2 (COVID-19) Spike RBD with orb1239994 at 20 ug/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green) and DAPI staining (blue). |
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Overexpression Validation in Spike Transfected 293 Cells. Loading: 10 µg per lane of 293 cell lysate. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD, orb1239994 (4 µg/mL), 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: WT 293 cells and Lane 2: SARS-CoV-2 Spike overexpressed 293 cells. |
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Western Blot Validation with SARS-CoV-2 (COVID-19) Spike RBD+SD1 Recombinant Protein. Loading: 30 ng per lane of SARS-CoV-2 (COVID-19) Spike RBD+SD1 recombinant protein, orb1231587. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD, orb1239994, 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: 1 µg/mL and Lane 2: 2 µg/mL. |
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ELISA Validation with SARS-CoV-2 (COVID-19) Spike RBD+SD1 Recombinant Protein. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD antibody, orb1239994 (1 µg/mL). A direct ELISA was performed using SARS-CoV-2 (COVID-19) Spike RBD+SD1 recombinant protein (orb1231587) as coating antigen and the anti-SARS-CoV-2 (COVID-19) Spike RBD antibody as the capture antibody. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:20000 dilution. Detection range is from 8 ng/mL to 1000 ng/mL. |
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Western Blot Validation with SARS-CoV-2 (COVID-19) Spike RBD+SD1+SD2 Recombinant Protein. Loading: 30 ng per lane of SARS-CoV-2 (COVID-19) Spike RBD+SD1+SD2 recombinant protein, orb1231586. Antibodies: SARS-CoV-2 (COVID-19) Spike RBD, orb1239994, 1 h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: 1 µg/mL and Lane 2: 2 µg/mL. |