By analyzing the amino acid sequence of the SARS-CoV-2 strain (MN908947), 20-mer peptides with an overlap of 10 aa residues, partially covering four structural proteins (S, N, M, E) of SARS-CoV-2, were chemically synthesized by GenScript (Jiangsu, China), and ultimately yielded 136 peptides. We also purchased the RBD protein (GenScript, Jiangsu, China) and N protein (VACURE Biotechnology, Sichuan, China) of SARS-CoV-2 and set them as protein probes in the experiments.
Rbd protein
Manufactured by GenScript
Sourced in United States
The RBD protein is a laboratory reagent used for research and development purposes. It is a recombinant protein derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The RBD protein is used in various applications, including assay development, antibody testing, and vaccine research and development.
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4 protocols using rbd protein
1
SARS-CoV-2 Structural Protein Peptides
2
SARS-CoV-2 Spike Antibody Generation
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Mice (BALB/c) were immunized
with the synthetic sequence of SARS-CoV-2
Spike DNA on days 0 and 14 by intramuscular immunization followed
by subcutaneous delivery of recombinant RBD protein (Genscript, USA)
on day 28, as described.23 (link),32 (link) One week after the
final immunization, the immune sera from the immunized mice were collected
and evaluated by enzyme-linked immunosorbent assay (ELISA) to detect
the presence of antibodies targeting SARS-CoV-2 Spike. After confirmation,
mouse splenocytes were used to generate hybridomas as described previously.33 (link) Subsequently, positive hybridoma clones were
characterized by an ELISA, and those selected were further subcloned
and expanded. The antibodies were purified from hybridoma supernatants
and used for further studies.
with the synthetic sequence of SARS-CoV-2
Spike DNA on days 0 and 14 by intramuscular immunization followed
by subcutaneous delivery of recombinant RBD protein (Genscript, USA)
on day 28, as described.23 (link),32 (link) One week after the
final immunization, the immune sera from the immunized mice were collected
and evaluated by enzyme-linked immunosorbent assay (ELISA) to detect
the presence of antibodies targeting SARS-CoV-2 Spike. After confirmation,
mouse splenocytes were used to generate hybridomas as described previously.33 (link) Subsequently, positive hybridoma clones were
characterized by an ELISA, and those selected were further subcloned
and expanded. The antibodies were purified from hybridoma supernatants
and used for further studies.
3
ELISA Protocol for SARS-CoV-2 RBD Antibody Detection
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In short, microwells of ELISA plates (Corning, NY, USA) were coated with 50 ng RBD protein each (Genscript, Nanjing, China) at 4 °C overnight. Plates were blocked with 10% FBS in PBS for 1 h at 37 °C, and then incubated with 2-fold serially diluted serum samples for another hour. After washing three times, plates were incubated with rabbit anti-mouse IgG-HRP antibody at a dilution of 1:20,000 (Abcam, Cambridge, UK) at 37 °C for 1 h. Tetramethylbenzidine (Solarbio, Beijing, China) and hydrogen peroxide were used for color development, and the reaction was stopped with 2 M H2SO4. The absorbance was measured at 450 nm, and an optical density at 450 nm (OD450) value greater than 2.1-fold of the background value was regarded as positive [25 (link)].
4
SARS-CoV-2 and Influenza Vaccine Proteins
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The PR8 HA recombinant protein and the SARS-CoV-2 RBD protein used for vaccine studies were purchased from Sino Biological.
The PR8 HA and RBD proteins used for generating fluorescently-labeled probes for B cell analyses and PR8 HA ELISA studies were produced as described previously (Amanat et al., 2020 (link); Margine et al., 2013 (link); Stadlbauer et al., 2020 (link)). The RBD protein used for ELISA was purchased from GenScript.
The PR8 HA and RBD proteins used for generating fluorescently-labeled probes for B cell analyses and PR8 HA ELISA studies were produced as described previously (Amanat et al., 2020 (link); Margine et al., 2013 (link); Stadlbauer et al., 2020 (link)). The RBD protein used for ELISA was purchased from GenScript.
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