Mouse Monoclonal Antibody to SARS-CoV2 S-Protein ACE2 Binding Domain Cat# MCA-2G1

      In late 2019 a novel infectious disease was discovered in Wuhan, China which was quickly recognized to be caused by a previously unknown RNA coronavirus. The virus was very rapidly isolated, the full RNA sequence determined and put on-line on the 10th of January 2020. The sequence revealed that the virus was most closely related to certain bat coronaviruses and the severe acute respiratory syndrome (SARS) coronavirus. Immediately biotechnology companies and research institutes used the RNA sequence information to generate vaccine candidates. The SARS virus was known to enter and infect human cells by means of the so-called spike or S-protein which binds to the extracellular domain of the angiotensin converting enzyme 2 (ACE2) protein, which is then internalized bringing the virus into the cell. Cryoelectron microscopy and binding studies quickly determined that the S-protein of SARS-CoV2 is structurally similar to to that of the SARS virus and also binds to the ACE2 receptor, albeit with higher affinity than the S-protein of SARS. This focuses attention on the ACE2 binding site on the SARS-CoV2 S-protein and for the complementary region on ACE2 which binds the SARS-CoV2 S-protein. We therefore expressed both these regions in E. coli, our products PROT-R-SARS-CoV2-bd and PROT-R-ACE2-bd and raised antibodies to them.

Vero cells are a cell line derived derived from monkey kidney and express an ACE2 receptor similar to that found in humans, and are therefore susceptible to infection by patient derived SARS-CoV2 virus. The cells were grown in culture, fixed, and stained with the DNA dye DAPI, revealing cell nuclei (control, top left), The remaining three panels show staining of similar cells following infection with SARS-CoV2 virus. Cells were then reacted with all three EnCor antibodies to the cell binding domain of the SARS-CoV2 spike protein, namely MCA-2G1, MCA-5G8 and RPCA-SARS-CoV2-bd as indicated. Antibody binding was revealed with appropriate ALEXA green (top right, bottom left) or red (bottom right) secondary antibodies. The virus accumulates in the cytoplasm of infected cells. The cells were grown and infected under appropriate safety and containment in the University of Florida Department of Pathology. Mouse select image above for larger view.



Immunofluorescent analysis of transfected HEK293 cells with the SARS-CoV2-bd-construct under high magnification, stained with mouse mAb to SARS-CoV2-bd, MCA-2G1, dilution 1:1,000, in green. Cells were costained with rabbit pAb to HSP60, RPCA-HSP60, dilution 1:2,000, in red. The blue is Hoechst staining of nuclear DNA. MCA-2G1 antibody reveals overexpression of SARS-CoV2-bd protein presumanbly in lysosomes only in transfected cells, while the HSP60 antibody labels mitochondria in all cells. Mouse select image above left for larger view.

This antibody was raised against a recombinant construct of the SARS-CoV-2 spike or S-protein which includes the entire region which interacts with ACE2. The specific binding to ACE2 is essential for viral internalization and infection. We designed this construct based on amino acids 308-541 in the S-protein sequence in Isolate Wuhan-Hu-1, complete genome. This is a defined globular domain recently shown to include all of the amino acids necessary for ACE2 binding. The construct was expressed in and purified from E. coli and includes an N-terminal His-tag and other vector derived sequence shown underlined below. Amino acids which interact directly with the ACE2 protein are printed in bold, data based on the cryoEM study of Walls et al. (3).


MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMADIGS EFVEKGIYQT  60
SNFRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS 120
TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA 180
WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY 240
GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNF 286


Number of amino acids: 286
Molecular weight: 32074.01
Theoretical pI: 8.01

Amino acid composition:
Ala (A) 17 5.9%
Arg (R) 13 4.5%
Asn (N) 22 7.7%
Asp (D) 16 5.6%
Cys (C) 9 3.1%
Gln (Q) 9 3.1%
Glu (E) 11 3.8%
Gly (G) 21 7.3%
His (H) 8 2.8%
Ile (I) 11 3.8%
Leu (L) 16 5.6%
Lys (K) 16 5.6%
Met (M) 4 1.4%
Phe (F) 19 6.6%
Pro (P) 15 5.2%
Ser (S) 23 8.0%
Thr (T) 16 5.6%
Trp (W) 2 0.7%
Tyr (Y) 16 5.6%
Val (V) 22 7.7%

Total number of negatively charged residues (Asp + Glu): 27
Total number of positively charged residues (Arg + Lys): 29

Extinction coefficient 35340
Abs 0.1% (=1 g/l) 1.102, assuming all pairs of Cys residues form cystines

Extinction coefficient 34840
Abs 0.1% (=1 g/l) 1.086, assuming all Cys residues are reduced

      This antibody has not been tested on formalin fixed and paraffin embedded samples for IHC, and iso we cannot recommended it for this purpose.