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Abstract
Prompt COVID-19 diagnosis is urgently required to support infection control measures. Currently available serological tests for measuring SARS-CoV-2 antibodies use different target antigens, although their sensitivity and specificity presents a challenge. We aimed to develop an “in-house” serological ELISA to measure antibodies against SARS-CoV-2 by combining different protein antigens. Sera (n = 44) from COVID-19-confirmed patients were evaluated against different SARS-CoV-2 protein antigens and all potential combinations using ELISA. Patients’ sera were also evaluated against commercially available ELISA diagnostic kits. The mixture containing RBD 2.5 μg/mL, S2 1 μg/mL and N 1.5 μg/mL was found to be the most potent. Plates were incubated with patients’ sera (1:100), and goat anti-human alkaline phosphatase-conjugated IgG, ΙgM and IgA antibody was added. The cut-off value for each assay was determined using the mean optical density plus two standard deviations of pre-pandemic controls. The “in-house” ELISA displayed 91% sensitivity and 97% specificity for IgG antibodies, whereas its sensitivity and specificity for IgM and IgA were 75% and 95% and 73% and 91%, respectively. The “in-house” ELISA developed here combined three SARS-CoV-2 antigens (RBD, S2 and N) as capture antigens and displayed comparable and even higher sensitivity and specificity than otherwise quite reliable commercially available ELISA diagnostic kits.
Highlights
Coronavirus disease 2019 (COVID-19) is a significant infectious disease of respiratory distress declared as a pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1,2,3]
The assessment of specific antibodies against SARS-CoV-2, including both immunoglobulin G (IgG) and M (IgM), as well as A (IgA), which are induced quickly post-disease onset, offers an alternate highly sensitive and accurate diagnostic tool that may offset the limitations of real-time polymerase chain reaction (RT-PCR) [14]
We aimed to develop a serological approach of high sensitivity and specificity to promptly define a true positive SARS-CoV-2 infection by combining different protein antigens as capture antigens, considering that kinetics of antibodies against distinct protein antigens are not yet fully defined
Summary
Coronavirus disease 2019 (COVID-19) is a significant infectious disease of respiratory distress declared as a pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1,2,3]. The assessment of specific antibodies against SARS-CoV-2, including both immunoglobulin G (IgG) and M (IgM), as well as A (IgA), which are induced quickly post-disease onset, offers an alternate highly sensitive and accurate diagnostic tool that may offset the limitations of RT-PCR [14]. Several commercial serological kits for measuring SARS-CoV-2 IgG, IgA and IgM antibodies have been approved by the FDA [20] These assays target immunogenic coronavirus proteins, mainly the N protein, the S protein and the S1 fragment, which may differ significantly regarding its reactogenicity from the S protein as a whole, or the receptor-binding domain (RBD). We aimed to develop a serological approach of high sensitivity and specificity to promptly define a true positive SARS-CoV-2 infection by combining different protein antigens as capture antigens, considering that kinetics of antibodies against distinct protein antigens are not yet fully defined. Written informed consent was obtained from the guardians of all subjects
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