Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 17 3 2025 06 02 480 487 Sajjad Bahman Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Safar Farajnia Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Effat Alizadeh Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran Farzin Seirafi Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Hojjatollah Nozad Charoudeh Stem Cell Research Center and Department Applied Cell Sciences, Tabriz University of Medical Sciences, Tabriz, Iran Mohammad Kazem Hosseini Department Molecular Biology and Genetics, Faculty of Sciences, Istanbul University, Istanbul, Turkey 2024 07 07 2025 04 11 Background and Objectives: The aim of this study was to assess the effectiveness of a new linear epitope from the N-terminal domain (NTD) of the SARS-CoV-2 S protein in the diagnosis of COVID-19. Materials and Methods: Serum samples from patients were confirmed to have COVID-19 by means of RT-PCR. The linear epitope sequence of the NTD was amplified by RT-PCR, inserted into an expression vector, and produced in Escherichi coli (DE3) pLysS. Subsequently, the recombinant proteins were purified and refolded. The interaction between the purified protein and the antibodies in COVID-19 patient sera was evaluated using ELISA. Results: Sequencing verified that the N-terminal linear epitope was successfully cloned into the PET-22b vector with a 6His-tag at the C-terminal end. The presence of a 25 kDa band on SDS-PAGE indicated the successful purification of the recombinant protein using Ni-NTA chromatography. The results of ELISA showed that the NTD linear epitope had strong sensitivity (88%) and specificity (96%) for identifying viral infection in COVID-19 patients' blood samples. Conclusion: The findings of this study demonstrated that the NTD linear epitopes of the SARS-CoV-2 spike protein exhibit significant sensitivity and specificity for the diagnosis of COVID-19 infection using serological techniques. However, further evaluations involving larger sample sizes across diverse ethnic populations is essential. https://ijm.tums.ac.ir/index.php/ijm/article/view/4894 https://ijm.tums.ac.ir/index.php/ijm/article/download/4894/1790