Fabrication of a monoclonal antibody-based electro-immunosensor to detect SARS-CoV-2 nucleocapsid antigen

The nucleocapsid protein is one of the major structural proteins of the SARS-CoV-2 virus, which has caused the COVID-19 pandemic. Due to their high abundance in the host circulation system, post-early infection, and their participation in other cyclic events that support viral survival, they exert control over the viral life cycle within the host. Thus, assessing these protein levels in serum can serve as an early diagnostic tool for COVID-19 infections. In the current study, monoclonal antibodies (mAbs) targeting recombinantly produced NCoV2 (rNCoV2) antigen have been developed, characterized, and assessed for their immunodiagnostic potential by indirect ELISA, sandwich ELISA, and further integrated into electro-immunosensor platforms. Hybridomas secreting anti-rNCoV2 mAbs were obtained by fusion of mouse myeloma Sp2/0 cells with rNCoV2 antigen-immunized murine splenocytes using a chemical fusion agent. Hybridoma clones PAb8E8c3-G8 of isotype IgG2A and PBa1-A2 of isotype IgG1, both bearing ĸ light chains, were further developed and assessed for their diagnostic potential, as they have shown a stable growth pattern and the desired antibody response against the rNCoV2 antigen. The lowest concentration of antigen detected by both mAbs with confidence was 7.81 ng/well. Of the two, mAb PAb8E8c3-G8 was selected for mAb-based electro-immunosensor fabrication. A simple label-free GCE-carbon black-based electro-immunosensor was fabricated and enhanced with L-Cys/AuNP, onto which the mAb was immobilized. The integrated mAb electro-immunosensor could efficiently detect the rNCoV2 antigen, with LOD and LOQ values of 4.618 pg/mL and 15.24 pg/mL, respectively. Thus, in this study, we have generated mAbs against the rNCoV2 antigen, which have shown better diagnostic potential in terms of specificity and sensitivity.