Comparative evaluation of SARS-CoV-2 antigens as capture and detection elements in an in-house antigen-based ELISA...

The global SARS-CoV-2 pandemic highlighted the urgent demand for reliable serological assays to track infection and immune responses. Enzyme immunoassays (EIA) targeting viral antibodies provide a practical platform for quantifying antibody responses in humans and animals, serving as a framework for future pandemic preparedness and response.

This study aimed to develop and evaluate an in-house EIA for the detection of total antibodies against SARS-CoV-2 using recombinant antigens: Spike Protein (SP), Receptor Binding Domain (RBD), and Nucleocapsid Protein (NP).

Recombinant antigens were conjugated to horseradish peroxidase (HRP) for detection in a double-sandwich ELISA format. The optimized assay was compared with an electrochemiluminescence assay (ECLIA) to determine cut-off values via receiver operating characteristic (ROC) curve analysis. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden's J statistic were calculated. Assay optimization included testing dilutional linearity, incubation time, and reaction volume. The assay was applied to human and animal serum samples to assess versatility.

Among the antigens tested, the receptor binding domain (RBD) exhibited the highest signal intensity and demonstrated dilutional linearity, outperforming both the spike protein and nucleoprotein in assay sensitivity. The assay demonstrated a sensitivity of 85% and specificity of 100%, with a PPV of 100% and an NPV of 68%. The high Youden's J statistic (0.85) and clear separation between true positive and negative samples underscore the robustness of the RBD antigen in reliably detecting SARS-CoV-2-specific antibodies for serological diagnostics. The optimized EIA protocol allowed for a reduced incubation time without compromising assay performance, and increasing the reaction volume two-fold did not result in a significant gain in signal intensity.

The RBD-based EIA provides a reliable, efficient platform for COVID-19 serological surveillance, suitable for both human and animal testing, and supports broader applications in vaccine evaluation and One Health monitoring.