Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama.
COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since its first appearance in December of 2019, the COVID-19 pandemic has caused extremely high levels of mortality, morbidity, global economic breakdown, and consequent human suffering. While vaccination efforts are not extensive and rapid enough, the main tools to keep the virus under control are still keeping physical distancing, reinforce personal hygiene measures, using masks and early diagnosis of virus-infected persons, either symptomatic or not. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase-quantitative real-time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are extremely useful to measure the antibodies generated in humans after virus contact, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISAs assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor-binding domain. As a control, we also analyzed a group of prepandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few prepandemic samples displayed high OD values, suggesting the possibility of some crossreactivity. All four assays show very good repeatability, both intra-, and inter-assay; however, no clear relationship could be detected between positivity and time of sample collection for serology. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98-0.995). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24-0.95). Our results show that both antigens are very effective to reveal both specific IgG and IgM antibodies, with high sensitivity (range 0.929-0.99) and specificity (range 0.933-0.977). The estimated congruence with the RT-PCR test, as estimated by Cohen Kappa, indicates a high agreement in all cases (range 0.874-0.937). This test will allow health authorities to have a new tool to estimate seroprevalence and to manage and improve the serious sanitary situation created by this virus.
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