Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers
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” serologica...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-10-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/11/11/1970 |
| _version_ | 1797510691994730496 |
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| author | Theano Lagousi John Routsias Vana Spoulou |
| author_facet | Theano Lagousi John Routsias Vana Spoulou |
| author_sort | Theano Lagousi |
| collection | DOAJ |
| description | 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 (<i>n</i> = 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. |
| first_indexed | 2024-03-10T05:34:57Z |
| format | Article |
| id | doaj.art-f029449ab9494f3884ccbea4c8e15902 |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-10T05:34:57Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-f029449ab9494f3884ccbea4c8e159022023-11-22T23:00:27ZengMDPI AGDiagnostics2075-44182021-10-011111197010.3390/diagnostics11111970Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological BiomarkersTheano Lagousi0John Routsias1Vana Spoulou2First Department of Paediatrics, “Aghia Sophia” Children’s Hospital, Immunology and Vaccinology Research Laboratory and Infectious Diseases Department “MAKKA”, Athens Medical School, 11527 Athens, GreeceDepartment of Microbiology, Athens Medical School, 11527 Athens, GreeceFirst Department of Paediatrics, “Aghia Sophia” Children’s Hospital, Immunology and Vaccinology Research Laboratory and Infectious Diseases Department “MAKKA”, Athens Medical School, 11527 Athens, GreecePrompt 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 (<i>n</i> = 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.https://www.mdpi.com/2075-4418/11/11/1970SARS-CoV-2antibodiesserological tests |
| spellingShingle | Theano Lagousi John Routsias Vana Spoulou Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers Diagnostics SARS-CoV-2 antibodies serological tests |
| title | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers |
| title_full | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers |
| title_fullStr | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers |
| title_full_unstemmed | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers |
| title_short | Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Accurate and Prompt Coronavirus Disease 2019 (COVID-19) Diagnosis Using the Rational Selection of Serological Biomarkers |
| title_sort | development of an enzyme linked immunosorbent assay elisa for accurate and prompt coronavirus disease 2019 covid 19 diagnosis using the rational selection of serological biomarkers |
| topic | SARS-CoV-2 antibodies serological tests |
| url | https://www.mdpi.com/2075-4418/11/11/1970 |
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