Development of Monoclonal Antibodies against Severe Acute Respiratory Syndrome- Coronavirus-2 Nucleocapsid Protein for Antigen Detection

Abstract

Introduction: 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 worldwide, as it enabled necessary precautions for those exposed, thereby 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. Various antigen and antibody testing kits with varying sensitivities and specificities 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 the SARS-CoV-2 nucleocapsid protein, owing to its ability to express proteins in their native form. This was an experimental study that used SARS-CoV-2-confirmed clinical samples. RNA was extracted from the nasopharyngeal swab and subjected to target gene amplification using SARS-CoV-2 N gene primers. The amplicon was purified, and a recombinant antigen was generated using the heat-shock transformation method with a pEU-his-tagged vector (pEu-E01-HIS-MCS-TEV-N2). Sanger sequencing was performed to confirm the orientation of the recombinant antigen. Protein expression was performed using WGCFS, purified using affinity chromatography and confirmed using SDS-PAGE and western blotting. BALB/c mice were immunized using the purified His-tagged N antigen with an adjuvant. Booster immunization was performed after 2 weeks, and the spleen cells were harvested after another 2 weeks and fused with myeloma cells to form hybridomas. The hybridomas were screened and selected for sub-cloning. A single clone was used for monoclonal antibody (mAb) production. The generated mAb was screened for reactivity against recombinant and commercial antigens, clinical samples, the Beta and Omicron variants, and for specificity against other respiratory viruses. SARS-CoV-2 N protein was successfully expressed using WGCFS. The generated mAb demonstrated high reactivity against the recombinant antigen, the commercial antigen, and the 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 commercial (p=0.072) antigen. The mAb detected SARS-CoV-2 in clinical samples with CT values ranging from 15.45 to 31.87 and showed no cross-reactivity with other respiratory viruses, but exhibited minimal potential cross-reactivity with HSV-1/2. The study demonstrated the dependability of the N protein for diagnosis. The generated mAb was comparable to a commercial mAb and detected SAR-CoV-2 variants, suggesting recognition of conserved conformational epitopes and ensuring reliable detection. The mAb also detected clinical samples across a range of CT values in ELISA, indicating good sensitivity.