23MAk-S2RM-3C4

Mouse monoclonal antibody (cl. 3C4) to SARS-CoV-2 Spike glycoprotein Receptor Binding Domain (RBD)

Description: Mouse monoclonal antibody (clone 3C4) to SARS-CoV-2 Spike glycoprotein Receptor Binding Domain (RBD). Primary antibody, unconjugated.

Clone: 3C4

Host: mouse

Subclass: IgG1

Immunogen: SARS-CoV-2 Spike glycoprotein Receptor Binding Domain from mammalian HEK293 cells (Baltymas product code: 20-S2RBD-RHs-F).

Tested applications: ELISA, Western blot (WB), immunofluorescence (IF).

Image: SDS-PAGE and Western blot using monoclonal antibody 3C4 to SARS-CoV-2 Spike glycoprotein RBD (cat. code 23MAk-S2RM-3C4), 0.2 µg/ml, followed by secondary goat anti-mouse IgG antibody conjugated to HRP.

M – PageRuler™ Prestained Protein Ladder, 10 to 170 kDa, Thermo Fisher Scientific, 5 µl/well

1 – SCoV2-rS, SARS-CoV-2 Spike glycoprotein full-length trimeric ectodomain from mammalian HEK293 cells (20-S2S-THs-F), 1 µg/well

2 – SCoV2-RBD,  SARS-CoV-2 Spike glycoprotein Receptor Binding Domain from mammalian HEK293 cells (Baltymas product 20-S2RBD-RHs-F), 1 µg/well

3 – SCoV2-rS, SARS-CoV-2 recombinant Spike glycoprotein trimeric ectodomain from mammalian CHO cells (Baltymas product 20-S2S-TCg-F), 1 µg/well

4 – SCoV2-RBD,  SARS-CoV-2 Spike glycoprotein Receptor Binding Domain from mammalian CHO cells (Baltymas product 20-S2RBD-RCg-F), 1 µg/well

5 – HEK293 cell lysate (negative control), 1.5×10⁵ cells/well

6 – SCoV2-rN, SARS-CoV-2 nucleoprotein from S. cerevisiae (negative control; Baltymas product 20-S2N-ScB-F), 1 µg/well

Proteins were separated in 12% SDS-PAGE.

Blot was developed with TMB.

Graphs: ELISA titration curves.

ELISA titration curves using monoclonal antibody 3C4 to SARS-CoV-2 Spike glycoprotein RBD (Cat. code 23MAk-S2RM-3C4), followed by secondary anti-mouse antibody conjugated to HRP. 1 – SARS-CoV-2 Spike protein expressed in CHO cells (20-S2S-TCg-F), 2 – SARS-CoV-2 RBD protein expressed in CHO cells (20-S2RBD-RCg-F),

Apparent dissociation constants of 3C4:

SARS-CoV-2 Spike protein expressed in CHO cells (20-S2S-TCg-F) – 1,38 × 10^-8 ± 1,2 × 10^-9 M

SARS-CoV-2 RBD protein expressed in CHO cells (20-S2RBD-RCg-F) – 5,55 × 10^-9 ± 1,96 × 10^-10 M

ELISA titration curves using monoclonal antibody 3C4 to SARS-CoV-2 Spike glycoprotein RBD (Cat. code 23MAk-S2RM-3C4), followed by secondary anti-mouse antibody conjugated to HRP. 1 – SARS-CoV-2 Spike Alpha protein (B.1.1.7 variant) expressed in CHO cells (21-S2SUK1-TCg-F), 2 – SARS-CoV-2 Spike Beta protein (B.1.351 variant) expressed in CHO cells (21-S2SSA1-TCg-F), 3 – SARS-CoV-2 Spike Kappa protein (B.1.617.1 variant) expressed in CHO cells (21-S2SIN1-TCg-F), 4 – SARS-CoV-2 Spike Delta protein (B.1.617.2 variant) expressed in CHO cells (22-S2SDE1-TCg-F), 5 – SARS-CoV-2 Spike Omicron protein (B.1.1.529 variant) expressed in CHO cells (22-S2SOM1-TCg-F).

Apparent dissociation constants of 3C4 for mutant Spike protein variants:

SARS-CoV-2 Spike Alpha protein (B.1.1.7 variant) – 1,89 × 10^-8 M

SARS-CoV-2 Spike Beta protein (B.1.351 variant) – 4,6 × 10^-8 ± 2,32 × 10^-12 M

SARS-CoV-2 Spike Kappa protein (B.1.617.1 variant) –

SARS-CoV-2 Spike Delta protein (B.1.617.2 variant) – 3,7 × 10^-8 M

SARS-CoV-2 Spike Omicron protein (B.1.1.529 variant) – 1,31 × 10^-9 ± 6,26 × 10^-12 M

Recommended starting dilutions: WB 1/200-1/1000; ELISA 1/1000-1/10000. Optimal concentrations should be determined by the end user.

Immunofluorescence photos: Based on an indirect immunofluorescence test, monoclonal antibody 3C4 (10 µg/ml) recognizes ancestor strain SARS-CoV-2 virus in infected Vero E6 cells.

Uninfected cells (negative control)	Infected cells

Virus neutralization: Based on the surrogate neutralization test described in product 22-hACE2-DCg-G description sheet, monoclonal antibody 3C4 does not inhibit SARS-CoV-2 Spike glycoprotein (Baltymas products 20-S2S-TCg-G and 20-S2S-TCg-F) binding to hACE2 receptor (Baltymas products 22-hACE2-DCg-F and 22-hACE2-DCg-G).

Purity: Protein A purified.

Form: Sterile-filtered PBS, pH 7.2

Concentration: 1 mg/ml

Storage conditions: This product is stable at +2 – +8°C for 2-3 weeks. For a long-term storage the antibody can be stored at -20°C or -80°C (avoid freeze / thaw cycle). Alternatively, in 50% glycerol at -20°C.

Conditions of shipping: Blue ice (0-8°C). Shipment cost to European zone: 100 €.

Additional info: This antibody recognizes the SARS-CoV-2 Spike glycoprotein Receptor Binding Domain (RBD). The epitope recognised by the antibody is located  within aa 429-477 sequence of the Spike glycoprotein provided in UniProtKB sequence accession number: P0DTC2. The antibody reacts both with full-length Spike glycoprotein and with RBD.

Availability: can be prepared for shipment in 1 week.

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