Analytical Development and Optimisation of a Semi-in-House ELISA for the Detection of SARS-CoV-2 by using Spike and Nucleocapsid Protein Antigens

Introduction: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a betacoronavirus that emerged in late 2019, is the causative agent of Coronavirus Disease 2019 (COVID19). The viral spike (S) and Nucleocapsid Protein (NP) are key structural components and principal targets for antigen detection. Developing reliable in-house immunoassays targeting these proteins is essential to enhance laboratory capacity and reduce dependence on commercial diagnostic kits. Aim: To analytically develop and optimise semi-in-house direct and sandwich Enzyme Linked Immuno Sorbent Assay (ELISA) for individual and simultaneous detection of SARS-CoV-2 spike (S) and NP antigens. Materials and Methods: This was an analytical laboratorybased study conducted between April 2025 and October 2025 at King Fahd Medical Research Centre, King Abdulaziz University, Jeddah, Saudi Arabia. For direct ELISAs, assay conditions were optimised using checkerboard titration. Recombinant SARSCoV-2 S and/or NP antigens were used as positive controls, while MERS-CoV S and/or NP antigens were included to evaluate potential cross-reactivity. Horseradish Peroxidase (HRP)-conjugated monoclonal antibodies specific to SARSCoV-2 S and/or NP served as detection antibodies. Optimal antibody concentrations were defined as those producing the highest positive-to-negative Optical Density (OD) ratio, including blank controls. For sandwich ELISAs, plates were coated with monoclonal anti-SARS-CoV-2 antibodies targeting S/RBD and/ or NP as capture antibodies. Subsequent steps followed the optimised protocol established for the direct ELISA format. Analytical performance was evaluated by determining the assay cut-off value, Limit Of Detection (LOD), linear dynamic range, and intra- and inter-assay Coefficients of Variation (CV%). Cross-reactivity was assessed using MERS-CoV recombinant proteins and heat-inactivated virus preparations. Results: Direct ELISAs detected S antigen at ≥6.25 ng and NP antigen at ≥3.125 ng per well whereas sandwich ELISAs improved analytical sensitivity four-fold (LOD 0.78 ng). Detection using heat-inactivated virus demonstrated analytical sensitivity down to 0.049×105 TCID50/mL. Intra-assay CV ranged from 3.1- 6.1% and inter-assay CV from 6.2-9.2%. No cross-reactivity with MERS-CoV was observed under tested conditions. Conclusion: The developed semi-in-house ELISAs demonstrated acceptable analytical sensitivity and reproducibility for research applications. Clinical validation using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)-confirmed patient samples is required before diagnostic implementation.