GENERATION OF MONOCLONALANTIBODIES AGAINST SEVERE ACUTE RESPIRATORY SYNDROME - CORONAVIRUS-2 NUCLEOCAPSID PROTEIN FOR ANTIGEN DETECTION

Abstract

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent for COVID-19, is a positive-sense enveloped RNA virus belonging to the Betacoronavirus genus and is approximately 30 kilobases long. Since its emergence, SARS-CoV-2 has resulted in over 700 million cases and millions of deaths worldwide, highlighting the critical need for effective diagnostic tools to manage and control the spread of the virus. Testing for COVID-19 was the primary measure implemented by all governments globally, as it allowed for necessary precautions against those exposed, hence preventing community spread. Various methods are being used for SARS-CoV-2 diagnosis, with RT-PCR being the gold standard. Due to RT-PCR limitations, such as the need for qualified personnel and long turnaround time, point-of-care testing is important for mass testing. Different antigen and antibody testing kits with varying sensitivity and specificity have been developed and deployed for SARS-CoV-2 diagnosis. Developing highly sensitive and specific antigen detection kits requires monoclonal antibodies as they are reliable and highly specific. This study leveraged the wheat germ cell-free system (WGCFS) to express SARS-CoV-2 nucleocapsid protein, owing to its ability to express proteins in their native form. After successfully expressing and purifying His-tagged SARS-CoV-2 N protein using WGCFS, BALB/c mice were immunized, and hybridomas were generated and screened. A highly reactive hybridoma against the produced antigen was selected for mAb production. Indirect and sandwich ELISA were used to screen the reactivity of the monoclonal antibody against the recombinant antigen and commercial antigen. The mAbs were also assessed for their performance using RT-PCR confirmed positive samples with varying cycle threshold (CT) values, and their specificity was screened using intracellular fluid (ICF) of other respiratory viruses. The mAb demonstrated high reactivity against the recombinant antigen, commercial antigen, SARS-CoV-2 Beta and Omicron variants. There was no significant difference in the binding affinity of the mAb and commercial mAb against the recombinant (p=0.12) and a commercial (p=0.072) antigen. The mAb detected SARS-CoV-2 from clinical samples with varying CT values ranging from 15.45 to 31.87 and exhibited no cross-reactivity against other respiratory viruses. This study findings highlight that the mAb can be used to detect antigens against SARS-CoV-2 for diagnosis and surveillance.