Abstract
To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2.
Highlights
Coronavirus Disease 2019 (COVID-19) is a novel acute respiratory disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1,2]
We construct two clones based on pCDNA3.1 vector to express receptor-binding domain (RBD) and S protein with two stabilizing proline mutation in the C-terminal S2 fusion machinery according to previous report using 293F cells (Figure 1A,B) [4]
A recent study had collected six commercially available SARSCoV-associated antibodies and one monoclonal antibody against SARS-CoV-2 S to test whether they could be used in SARS-CoV-2 related immune detection; there were only two antibodies could stain SARS-CoV-2 positive formalin-fixed and paraffin-embedded (FFPE) cell pellets in IHC and IFA assay, including a rabbit polyclonal antibody against S protein of SARS-CoV and a mouse monoclonal antibody against NP protein of SARS-CoV [15]
Summary
Coronavirus Disease 2019 (COVID-19) is a novel acute respiratory disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1,2]. The amino acid sequence homology between severe acute respiratory syndrome coronavirus-related coronavirus (SARS-CoV) and SASR-CoV-2 approximates to 76% for the spike (S) proteins [3]. SARS-CoV-2 and SARSCoV share a common host-cell receptor protein, angiotensin converting enzyme 2 (ACE2), interacted by S protein for entry into the target cell [6,7,8]. The receptor-binding domain (RBD) on S1 subunit plays a key role in binding to receptor on the surface of host cell and becomes an important target for functional study and vaccine and therapeutics development [9]. It is essential to develop a monoclonal antibody (mAb) that recognizes linear epitope on RBD, which will be useful for the RBD detection
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