{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T00:00:33Z","timestamp":1789084833463,"version":"build-2803163510"},"reference-count":28,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,1,11]],"date-time":"2021-01-11T00:00:00Z","timestamp":1610323200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Determining the presence of antibodies in serum is important for epidemiological studies, to be able to confirm whether a person has been infected, predicting risks of them getting sick and spreading the disease. During the ongoing pandemic of COVID-19, a positive serological test result can suggest if it is safe to return to work and re-engage in social activities. Despite a multitude of emerging tests, the quality of respective data often remains ambiguous, yielding a significant fraction of false positive results. The human organism produces polyclonal antibodies specific to multiple viral proteins, so testing simultaneously for multiple antibodies appeared a practical approach for increasing test specificity. We analyzed immune response and testing potential for a spectrum of antigens derived from the spike and nucleocapsid proteins of SARS-CoV-2, developed a dual-antigen testing system in the ELISA format and designed a robust algorithm for data processing. Combining nucleocapsid protein and receptor-binding domain for analysis allowed us to completely eliminate false positive results in the tested cohort (achieving specificity within a 95% confidence interval of 97.2\u2013100%). We also tested samples collected from different households, and demonstrated differences in the immune response of COVID-19 patients and their family members; identifying, in particular, asymptomatic cases showing strong presence of studied antibodies, and cases showing none despite confirmed close contacts with the infected individuals.<\/jats:p>","DOI":"10.3390\/diagnostics11010102","type":"journal-article","created":{"date-parts":[[2021,1,11]],"date-time":"2021-01-11T11:36:11Z","timestamp":1610364971000},"page":"102","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Dual-Antigen System Allows Elimination of False Positive Results in COVID-19 Serological Testing"],"prefix":"10.3390","volume":"11","author":[{"given":"Andrei","family":"Komarov","sequence":"first","affiliation":[{"name":"VirIntel, LLC, Gaithersburg, MD 20877, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8968-029X","authenticated-orcid":false,"given":"Anna","family":"Kaznadzey","sequence":"additional","affiliation":[{"name":"VirIntel, LLC, Gaithersburg, MD 20877, USA"},{"name":"Institute for Information Transmission Problems, RAS, Bolshoy Karetny per. 19, 127051 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1191-9701","authenticated-orcid":false,"given":"Yue","family":"Li","sequence":"additional","affiliation":[{"name":"Health Informatics &amp; Data Science Georgetown University, Georgetown University Medical Center, 4000 Reservoir Road, NW, Washington, DC 20007, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0527-3182","authenticated-orcid":false,"given":"Maria","family":"Kireeva","sequence":"additional","affiliation":[{"name":"VirIntel, LLC, Gaithersburg, MD 20877, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ilya","family":"Mazo","sequence":"additional","affiliation":[{"name":"VirIntel, LLC, Gaithersburg, MD 20877, USA"},{"name":"Argentys Informatics, LLC, Gaithersburg, MD 20877, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,1,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Qian, Z., Dominguez, S.R., and Holmes, K.V. (2013). Role of the Spike Glycoprotein of Human Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Virus Entry and Syncytia Formation. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0076469"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1038\/s41586-020-2012-7","article-title":"A Pneumonia Outbreak Associated with a New Coronavirus of Probable Bat Origin","volume":"579","author":"Zhou","year":"2020","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1016\/j.cell.2020.02.052","article-title":"SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor","volume":"181","author":"Hoffmann","year":"2020","journal-title":"Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1038\/s41564-019-0392-y","article-title":"Antibody Responses to Viral Infections: A Structural Perspective across Three Different Enveloped Viruses","volume":"4","author":"Murin","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref_5","unstructured":"Baron, S. (1996). Medical Microbiology, University of Texas Medical Branch at Galveston. [4th ed.]."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Berkowitz, F.E., and Jerris, R.C. (2016). Practical Medical Microbiology for Clinicians, John Wiley & Sons.","DOI":"10.1002\/9781119066767"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1478","DOI":"10.3201\/eid2607.200841","article-title":"Severe Acute Respiratory Syndrome Coronavirus 2\u2212Specific Antibody Responses in Coronavirus Disease Patients","volume":"26","author":"Okba","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1038\/s41591-020-0897-1","article-title":"Antibody Responses to SARS-CoV-2 in Patients with COVID-19","volume":"26","author":"Long","year":"2020","journal-title":"Nat. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1200","DOI":"10.1038\/s41591-020-0965-6","article-title":"Clinical and Immunological Assessment of Asymptomatic SARS-CoV-2 Infections","volume":"26","author":"Long","year":"2020","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e245","DOI":"10.1016\/S1473-3099(20)30517-X","article-title":"Serology Testing in the COVID-19 Pandemic Response","volume":"20","author":"Peeling","year":"2020","journal-title":"Lancet Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Schmidt, S.B., Gr\u00fcter, L., Boltzmann, M., and Rollnik, J.D. (2020). Prevalence of Serum IgG Antibodies against SARS-CoV-2 among Clinic Staff. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0235417"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Bryant, J.E., Azman, A.S., Ferrari, M.J., Arnold, B.F., Boni, M.F., Boum, Y., Hayford, K., Luquero, F.J., Mina, M.J., and Rodriguez-Barraquer, I. (2020). Serology for SARS-CoV-2: Apprehensions, Opportunities, and the Path Forward. Sci. Immunol., 5.","DOI":"10.1126\/sciimmunol.abc6347"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1452","DOI":"10.1093\/infdis\/jiaa479","article-title":"SARS-CoV-2\u2013Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence","volume":"222","author":"Schepp","year":"2020","journal-title":"J. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1093\/ije\/25.6.1107","article-title":"Basic Methods for Sensitivity Analysis of Biases","volume":"25","author":"Greenland","year":"1996","journal-title":"Int. J. Epidemiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"W24","DOI":"10.1093\/nar\/gkx346","article-title":"BepiPred-2.0: Improving Sequence-Based B-Cell Epitope Prediction Using Conformational Epitopes","volume":"45","author":"Jespersen","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Klumpp-Thomas, C., Kalish, H., Drew, M., Hunsberger, S., Snead, K., Fay, M.P., Mehalko, J., Shunmugavel, A., Wall, V., and Frank, P. (2020). Standardization of Enzyme-Linked Immunosorbent Assays for Serosurveys of the SARS-CoV-2 Pandemic Using Clinical and at-Home Blood Sampling. MedRxiv, Preprint.","DOI":"10.1101\/2020.05.21.20109280"},{"key":"ref_17","first-page":"101","article-title":"Interval Estimation for a Binomial Proportion","volume":"16","author":"Brown","year":"1999","journal-title":"Stat. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1007\/BF00994018","article-title":"Support-Vector Networks","volume":"20","author":"Cortes","year":"1995","journal-title":"Mach. Learn."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1007\/s10462-017-9611-1","article-title":"Selecting Training Sets for Support Vector Machines: A Review","volume":"52","author":"Nalepa","year":"2019","journal-title":"Artif. Intell. Rev."},{"key":"ref_20","first-page":"2825","article-title":"Scikit-Learn: Machine Learning in Python","volume":"12","author":"Pedregosa","year":"2011","journal-title":"J. Learn. Res."},{"key":"ref_21","first-page":"1717","article-title":"Detection of Antibodies against SARS-Coronavirus Using Recombinant Truncated Nucleocapsid Proteins by ELISA","volume":"18","author":"Lee","year":"2008","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1016\/j.jviromet.2011.07.005","article-title":"Development of a Recombinant Truncated Nucleocapsid Protein Based Immunoassay for Detection of Antibodies against Human Coronavirus OC43","volume":"177","author":"Blanchard","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"956","DOI":"10.1126\/science.abc7520","article-title":"Isolation of Potent SARS-CoV-2 Neutralizing Antibodies and Protection from Disease in a Small Animal Model","volume":"369","author":"Rogers","year":"2020","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1038\/s41586-020-2571-7","article-title":"Potent Neutralizing Antibodies against Multiple Epitopes on SARS-CoV-2 Spike","volume":"584","author":"Liu","year":"2020","journal-title":"Nature"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.cell.2020.05.025","article-title":"Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients\u2019 B Cells","volume":"182","author":"Cao","year":"2020","journal-title":"Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1038\/s41586-020-2548-6","article-title":"Potently Neutralizing and Protective Human Antibodies against SARS-CoV-2","volume":"584","author":"Zost","year":"2020","journal-title":"Nature"},{"key":"ref_27","first-page":"5","article-title":"Beware of False-Positive Results with SARS-CoV-2 Antibody Tests","volume":"102","author":"Ebell","year":"2020","journal-title":"AFP"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"m3325","DOI":"10.1136\/bmj.m3325","article-title":"Testing for SARS-CoV-2 Antibodies","volume":"370","author":"Watson","year":"2020","journal-title":"BMJ"}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/11\/1\/102\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:09:39Z","timestamp":1760159379000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/11\/1\/102"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,1,11]]},"references-count":28,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,1]]}},"alternative-id":["diagnostics11010102"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics11010102","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,1,11]]}}}