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Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. −80°C, −20°C and initially at 4°C followed by −80°C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. The ELISA system showed the highest sensitivity (92.9%) for samples with Ct ≤30 and preserving at −80°C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.

Detection of SARS-CoV-2 by antigen ELISA test is highly swayed by viral load and sample storage condition