DEVELOPMENT OF RECOMBINANT MONOCLONAL ANTIBODIES

Abstract

The global pandemic which spread throughout the world in 2019, known as COVID-19 is caused by SARS-CoV-2 virus infection. Fever, dry cough, exhaustion, and acute respiratory distress syndrome are among the clinical symptoms of this disease, which can lead to the death of the infected patient. COVID-19 is currently one of the main priorities, for scientists to develop novel strategies. In the past, immunotherapy in the form of vaccines and antibodies has proven their efficacy against viral infectious illnesses. Antibody-based therapies are being developed quickly by researchers to inhibit and/or neutralize the coronavirus in infected patients. As opposed to vaccines, monoclonal antibodies (mAbs), are a type of passive immunotherapy that can be used as effective therapeutics for various diseases. Furthermore, since monoclonal antibodies can be developed in a lab or can be isolated from the infected person’s blood, they are much more precise, safe, and specific than convalescent plasma therapy. In contrast to convalescent plasma, which is composed of polyclonal antibodies in patient-derived serum, monoclonal antibodies have specific activity against a predefined target. It has been demonstrated that anti-SARS-CoV-2 monoclonal antibodies against the spike protein of SARS-CoV-2 are clinically effective in treating Covid-19 infection.

In this proposal, we have used spike protein of SARS-Cov-2 virus as an antigen to immunize wild-type Balb/c mice. We harvested the splenocytes of the immunized mice to prepare hybridomas by fusing them with myeloma cells; which were further sub-cloned by limiting dilution method to produce monoclonal antibodies. Thereafter, we performed molecular characterization of these monoclonal antibodies. Briefly, approx. 1-2 million antibody- secreting hybridoma cells were collected and homogenized using an RNase-free 5 ml syringe. The RNeasy Mini Spin kit was utilized to isolate total RNA. cDNA was synthesized using Oligo-dT primers, Random Hexamers, RNA template, and Reverse Transcriptase, at 42°C for 1 hour. The reverse transcriptase was inactivated by incubation at 70°C for 15 minutes. The resulting cDNA was used as a template for Nested PCR in association with touchdown PCR to amplify the DNA using various 5′ and 3′ primers specific for Variable and Constant region of heavy chain in one reaction and for light chain in another reaction. Thereafter, amplified DNA fragments were purified using PCR Purification Kit and were examined on 1% agarose gel. These PCR purified DNA fragments were ligated into pGEM-T-EAsy vector and were transformed into competent E. coli DH5α cells. After transformation, the cells were plated onto LB Agar/Amp/Xgal/IPTG plates to identify colonies that successfully transformed. Once the colonies were obtained, plasmid DNA was isolated using Plasmid DNA Extraction Mini Kit. The plasmid DNA was subjected to restriction digestion using EcoR1 HF enzyme, which cuts the plasmid flanking the MCS. To confirm the presence of expected size insert, the digestions were examined on an agarose gel. The mAbs are likely to contribute to a decrease in viral load by blocking SARS-CoV2 ability to bind to host cell receptors and preventing virus attachment to cell surface receptors, depriving SARS-CoV-2 of its ability to infect host cells. The monoclonal antibodies against SARS-CoV-2 virus have the potential to be employed for both infection prevention and treatment.