Abstract
The early and accurate diagnosis of COVID-19 is essential for efficient disease management. Traditionally, enzyme-linked immunosorbent assays for detecting IgG antibodies to SARS-CoV-2 have relied on single antigens, such as the spike or nucleoprotein. However, the test sensitivity has not been satisfactory. This study diverges from conventional approaches by developing an indirect ELISA assay utilizing a novel, highly sensitive fusion antigen incorporating both the receptor-binding domain (RBD) and nucleoprotein (N). The physicochemical characteristics of this unique antigen were examined and confirmed through experimental validation in our recent research. Our primary objective is to enhance the diagnostic sensitivity and accuracy of the ELISA assay. The indirect ELISA assay was developed using a novel fusion antigen incorporating both the RBD and nucleoprotein (N) of SARS-CoV-2. A cohort of 112 patients presenting with COVID-19 symptoms was evaluated to detect SARS-CoV-2 RNA. Additionally, 25 serum samples from healthy individuals were selected as the negative control group. The study was conducted in Tehran Province, Iran, from March to July 2022. Serum samples from patients who tested positive using the real-time PCR method were collected, and an in-house indirect ELISA assay was developed. The sensitivity and specificity of the assay were evaluated and compared with the results of the commercial Euroimmun anti-SARS-CoV-2 IgG assay, which served as the gold standard. The ROC curve was analyzed using GraphPad Prism to assess the accuracy and reliability of the in-house ELISA assay. The in-house ELISA assay developed in this study demonstrated successful performance and showed 100% sensitivity and 96% specificity in detecting anti-SARS-CoV-2 IgG antibodies. Receiver operating characteristic (ROC) analysis yielded an area under the curve (AUC) of 0.976, indicating high accuracy. Our in-house ELISA assay exhibits high sensitivity and specificity, underscoring its suitability for commercial development as a reliable diagnostic kit for detecting COVID-19 cases.