Currently there are few specific antibodies that can differentiate SARS-CoV-2 from other coronaviruses. Haripriya Sridharan, Swathi SundarRaj, Pavitra Desai and Thangaraj Karuppuchamy discuss antibodies for SARS-CoV-2 detection and research.
The need for SARS-CoV-2 specific antibodies
As the SARS-CoV-2 crisis has gripped the world, science is racing at breakneck speed to understand this disease from all angles to come up with better preventive, diagnostic or treatment options. There is an immediate and urgent need for many different types of reagents that will aid this research. Antibodies are some of the most routinely used and versatile tools in biological research and can be used in a variety of assays, making them the “go to” reagents for protein-based detection. Antibodies have been in the limelight for their neutralization properties, and several groups have reported the isolation of neutralizing SARS-CoV-2 antibodies from convalescing patients. Antibodies are important research reagents of infection discovery that can be incorporated into simple to use point-of-care detection kits such as lateral flow or other research immunoassays. In addition to their diagnostic and research value, antibodies are also important reagents that aid fundamental research to understand the biology of SARS-CoV-2 infections.
Most of the currently available SARS-CoV-2 antibodies in the market have actually been developed to detect other coronavirus members such as SARS-CoV or MERS and have since been found to cross react with SARS-CoV-2 due to recognition of conserved regions of the structural proteins. However, due to the time sensitive nature of this disease and the emergence of the virus, there are few SARS-CoV-2 specific antibodies that can differentiate SARS-CoV-2 from other coronaviruses. To address this immediate need, we have co-developed SARS-CoV-2 specific recombinant monoclonal antibodies with a leading pharmaceutical company. Recombinant monoclonal antibodies offer advantages over polyclonal or hybridoma derived monoclonal antibodies because they offer lot to lot reproducibility and can be manufactured in an animal origin free production system.
Development of SARS-CoV-2 specific antibodies
To develop antibodies against SARS-CoV-2, we have focused on the spike protein of SARS-CoV-2, which is a trimeric complex molecule present in the viral membrane. The receptor binding domain (RBD) of the spike binds to cellular receptor ACE2 and this interaction is crucial for the internalization of the virion leading to subsequent infection. Consequently, the RBD-ACE2 interaction has been a hot relevant target for development of antivirals and neutralizing antibodies.
To develop antibodies against SARS-CoV-2, we started with a scFv (single chain fragment variable) library generated by computational expansion of published antibody structures against SARS-CoV, from which we selected several specificities for SARS-CoV-2. Development of these antibodies included grafting each specificity onto a human IgG1 backbone and a rabbit IgG antibody backbone, to produce fully humanized and human Fab-Rabbit Fc chimeric antibodies. The choice of the different backbones is to offer certain flexibility to researchers to use these antibodies in their specific applications. Human antibodies are useful as controls for development of kits to detect and characterize immune responses to SARS-CoV-2 and can also be used in the development of neutralization kits, while rabbit antibodies are routinely used in many research laboratories. Figure 1 shows the representation of the two backbones used.
Figure 1: Schematic representation of the SARS-CoV-2 antibody backbones. A. Fully human backbone B. human Fab and rabbit Fc chimeric backbone. Black represents human sequences and red represents rabbit sequences
The antibodies were purified from ExpiCHO™ Expression System, which is an animal origin free system to generate recombinant antibodies and were tested in a variety of applications. Three different specificities in the two backbones described above have been developed and tested in a variety of applications such as indirect ELISA, immunofluorescence, and flow cytometry. Table 1 summarizes the list of antibodies launched. Figure 2 shows the application testing for one of these antibodies, Anti-SARS-CoV-2 Spike Protein (RBD) Chimeric Recombinant Rabbit Monoclonal Antibody (P05DHuRb) (Product #703971).
Table 1: List of launched Recombinant Human and Human-Rabbit Chimeric antibodies for SARS-CoV-2 Spike Protein (RBD)
Figure 2: Application testing for Anti-SARS-CoV-2 Spike Protein (RBD) Chimeric Recombinant Rabbit Monoclonal Antibody (P05DHuRb) (Product #703971). A. Indirect ELISA using purified Spike protein (RBD) from SARS-CoV-2. B. Flow cytometry using HEK 293 cells transfected with SARS-CoV-2 spike protein (pink histogram- Transfected cells; blue histogram- untransfected control cells). C. Immunofluorescence using HEK 293 cells transfected with SARS-CoV-2 spike protein. D. Untransfected control. Green represents spike protein, red represents F Actin, and blue represents nuclei.
Antibody specificity to SARS-CoV-2 spike protein
One of the important goals for this project was to establish antibody specificity for spike protein from SARS-CoV-2, because the RBD of spike proteins from SARS CoV and SARS-CoV-2 share 73% sequence similarity. In a bead based ELISA panel containing antigens from SARS-CoV-2, SARS CoV, MERS and other human coronaviruses, all six human and chimeric antibodies specifically recognize the Spike protein from SARS-CoV-2 but not those from other coronaviruses. This demonstrates that these antibodies are specific using relative expression testing. Figure 3 represents the specificity data for one of these antibodies, the Anti-SARS-CoV-2 Spike Protein (RBD) Recombinant Human Monoclonal Antibody (T01KHu) (Product # 703958).
Figure 3: Anti-SARS-CoV-2 Spike Protein (RBD) Recombinant Human Monoclonal Antibody (T01KHu) (Product # 703958) only recognizes Spike protein of SARS-CoV-2 (SARS-CoV-2 S, SARS-CoV-2 RBD, and SARS-CoV-2 S1). Antibody specificity was demonstrated by bead- based Immunoassay showing differential detection of target antigen across a panel of related viral proteins.
Blocking RBD-ACE2 interaction
We then wanted to understand if any of these antibodies are able to block spike protein RBD binding to the ACE2 receptor. Utilizing a competitive ELISA, we were able to demonstrate the blocking properties for four of these antibodies. Figure 4 represents the neutralization data for four antibodies (Product #s 703958, 703959, 703973, and 703974), further demonstrating specificity of these antibodies to the target using neutralization verification.
Figure 4: Spike protein RBD-ACE2 interaction blocking by Anti-SARS-CoV-2 Recombinant Monoclonal Antibodies. ELISA based SARS-CoV-2 Inhibitor Screening Assay shows that binding of SARS-CoV-2 Spike Protein RBD to Human ACE2 receptor was inhibited in the presence of indicated antibodies.
Antibodies for SARS-CoV-2 Detection and Research
To address an important market demand for SARS-CoV-2 specific detection reagents, we have developed recombinant monoclonal antibodies against SARS-CoV-2 spike protein RBD. These reagents are specific to SARS-CoV-2 and some of them are able to strongly inhibit the binding of spike protein to the ACE2 receptor. Keeping customer requirements in mind, we offer these antibodies in two different backbones in order to enable greater utility across applications. These reagents will be useful as scientists try to decode the biology of SARS-CoV-2 and develop robust assays to detect and neutralize SARS-CoV-2.
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For Research Use Only. Not for use in diagnostic procedures.