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Anti-SARS-CoV-2 antibodies can provide information on patient immunity, identify asymptomatic patients, and track the spread of COVID-19. Efforts have been made to develop methods to detect anti-SARS-CoV-2 antibodies in humans. Here, we describe a flow cytometric assay for the simultaneous detection of anti-SARS-CoV-2 IgG and IgM in human plasma. To assess the antibody response against the different SARS-CoV-2 structural proteins, five viral recombinant proteins, including spike protein subunit 1 (S1), N-terminal domain of S1 (S1A), spike receptor-binding domain (RBD), spike protein subunit 2 (S2), and nucleocapsid protein (N), were generated. A comparison of the antibody profiles detected by the assay with plasma from 100 healthy blood donors collected prior to the COVID-19 pandemic and plasma from 100 virologically confirmed COVID-19 patients demonstrated a clear discrimination between the two groups. Among the COVID-19 patients, the antibody responses for the viral proteins, as determined by their prevalence, were anti-RBD IgG\u2009=\u2009anti-N IgG\u2009&gt;\u2009anti-S1 IgG\u2009&gt;\u2009anti-S1A IgG\u2009&gt;\u2009anti-S2 IgG, and anti-RBD IgM\u2009&gt;\u2009anti-S1 IgM\u2009&gt;\u2009anti-N IgM\u2009&gt;\u2009anti-S2 IgM. The prevalence of anti-SARS-CoV-2 IgG and IgM was not associated with sex, age, race, days after the onset of symptoms, or severity of illness, except for a higher prevalence of anti-S2 IgG being observed in men than in women. The levels of anti-RBD IgG were higher in patients 65\u00a0years and older and in patients who had severe symptoms. Similarly, patients who had severe symptoms exhibited higher levels of anti-S1 and anti-S1A IgG than patients who had mild or moderate symptoms. The levels of anti-RBD IgM tended to be higher in men but did not differ among age, race, days after the onset of symptoms, or severity of illness. Our study indicates that the flow cytometric assay, especially using RBD as target antigen, can be used to detect simultaneously anti-SARS-CoV-2 IgG and IgM antibodies in human plasma.<\/jats:p>","DOI":"10.1038\/s41598-025-92389-8","type":"journal-article","created":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T01:52:23Z","timestamp":1742953943000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Flow cytometric analysis of the SARS coronavirus 2 antibodies in human plasma"],"prefix":"10.1038","volume":"15","author":[{"given":"Jia-Long","family":"Fang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leeza","family":"Shrestha","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frederick A.","family":"Beland","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,3,25]]},"reference":[{"key":"92389_CR1","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1186\/s12985-019-1182-0","volume":"16","author":"D Schoeman","year":"2019","unstructured":"Schoeman, D. & Fielding, B. 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