en <p>Background: SARS-CoV-2 is a novel coronavirus that has caused dramatic loss of life and poses an unprecedented public health challenge worldwide. The nucleocapsid (N) protein of SARS-CoV-2 is the most abundant viral protein and a potent immunogen. Methods: In the current study, the N gene of SARS-CoV-2 was amplified from RNA extracted from a COVID-19 positive patient and then cloned into the pCold-I expression vector. The full-length His-tagged N protein was expressed in Escherichia coli (E. coli) using 0.5 mM IPTG and subsequently purified by nickel affinity chromatography. The purified N protein was characterized using sodium dodecyl sulfate&ndash;polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Furthermore, the purified N protein was applied in SARS-CoV-2 IgG and IgM ELISA immunoassays. Results: The results showed that purification of the N protein in the presence of urea yielded a protein band of approximately 48 kDa on SDS-PAGE, corresponding to the full-length N protein. Additionally, Western blot analysis of the purified recombinant N protein showed a band of the same molecular weight. In the SARS-CoV-2 IgG ELISA assay, anti-N protein antibodies from a COVID-19 positive patient&rsquo;s serum successfully recognized the coated N protein. In the IgM ELISA test, an N-HRP conjugate was used in ELISA wells to reveal the interaction of HRP-conjugated N protein with pre-coated anti-N protein antibodies (IgM isotype). Conclusion: These results indicate that the expressed N protein of SARS-CoV-2 could serve as a valuable reagent for the development of antibody-based immunoassays to detect SARS-CoV-2 IgG and IgM antibodies.</p> ELISA, Nucleocapsid (N) protein, Recombinant protein expression, SARS-CoV-2, Serological diagnosis 55 60 https://www.ajmb.org/En/Article.aspx?id=70638 https://www.ajmb.org/PDF/En/FullText/70638.pdf SaeidehZamani KoukhalooMonoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran92456FarshidMoosaviMonoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran92369BaharehZamaniMonoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran92367NiloofarAgharezaeeMonoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran92368ParisaYousefiMonoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran92371MahboobehNazari11474JafarMahmoudian114