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The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01–75.12) and 100% (95% CI: 97.57–100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17–96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19–88.71) for samples with Ct values < 30. Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus. [Display omitted] • Antigen-based serological assays are efficient methods for SARS-CoV-2 screening. • The viral nucleoprotein is the best target due to protein abundance and stability. • We designed a sandwich ELISA using novel nucleoprotein specific MAbs. • Antigen titer correlates inversely to cycle threshold values in clinical samples. • This assay is sensitive and highly specific irrespective of the viral variants. [ABSTRACT FROM AUTHOR]