Development of in-house, indirect ELISAs for the detection of SARS-CoV-2 spike protein-associated serology in COVID-19 patients in Panama

Abstract

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 first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. 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 useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA 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 analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. 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: 0.98–0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24–0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92–0.99), specificity (range 0.93–0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87–0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.

Development of Serological Assays and Seroprevalence Studies of the New Coronavirus 2019 (COVID-19): Reports from Saudi Arabia

“…At the international level, similar studies describing COVID-19 immunoassays have been reported from several research groups [ 29 , 30 , 31 , 32 , 33 , 34 ]. Most of these immunoassays were based on SARS-CoV-2 full-length S, NP, or fragments of S (e.g., receptor binding domain).…”

“…In some cases, assays were optimized not only for detection of IgM and IgG, but also for IgA [ 33 ]. Validation of results was also evaluated against RT-PCR, the MN assay, and commercially available kits [ 29 , 30 , 31 , 32 , 33 , 34 ]. Herein, we review several in-house immunoassays developed in Saudi Arabia.…”

Evaluation of S- and M-Proteins Expressed in Escherichia coli and HEK Cells for Serological Detection of Antibodies in Response to SARS-CoV-2 Infections and mRNA-Based Vaccinations

“…As E. coli RBD did not provide good results in the IgG-based ELISA, although it bound to the ACE2 receptor, it was not further considered for testing [24]. Thus, RBD was expressed in HEK cells, as previous reports indicated also a good sensitivity in ELISA [16,[25][26][27]. When the monomeric and dimeric versions of RBD expressed in HEK cells in-house and two commercial RBDs were coated in ELISA, the obtained OD 450 -values were identical within the error range (Figure S7).…”

Adaptation-proof SARS-CoV-2 vaccine design

“…All pre-pandemic serum samples remained unreactive. Some samples in the COVID-19 group showed low OD readouts similar to healthy samples, suggesting a lack of or a weak seroconversion 64,68,69 . Next steps may involve testing cross reactivity with patient samples of other coronaviruses.…”