{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T21:54:17Z","timestamp":1788472457473,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,9,8]],"date-time":"2025-09-08T00:00:00Z","timestamp":1757289600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Council Canada"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus requires enhanced biological containment facilities (i.e., CL3) to reduce the potential of laboratory infection to personnel and the spread of the virus into the community. While the use of an authentic live virus remains the gold standard for biological assays, alternative methods have been developed to effectively evaluate neutralization activity in the absence of a replicating viral agent. Here, we describe a cell-based spike\u2013ACE2 binding assay as a surrogate for neutralization of SARS-CoV-2 spike to identify potential neutralizing antibodies. A main advantage of this approach is the exclusion of infectious viral particles, increasing biosafety for laboratory personnel. The interaction of recombinant SARS-CoV-2 trimeric spike protein with ACE2 is monitored and quantified by flow cytometry. Notably, our previous studies have demonstrated the utility of this assay for other viruses, beyond SARS-CoV-2. The methodology presented here has exhibited a strong correlation to other widely accepted methods, such as pseudotyped lentiviral and live virus neutralization assays, in identifying neutralizing antibodies.<\/jats:p>","DOI":"10.3390\/mps8050104","type":"journal-article","created":{"date-parts":[[2025,9,8]],"date-time":"2025-09-08T11:51:12Z","timestamp":1757332272000},"page":"104","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["A Safe and Accessible Cell-Based Spike\u2013ACE2 Binding Assay for Evaluating SARS-CoV-2 Neutralization Activity in Biological Samples Using Flow Cytometry"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8902-5532","authenticated-orcid":false,"given":"Martin A.","family":"Rossotti","sequence":"first","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shannon","family":"Ryan","sequence":"additional","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9852-6865","authenticated-orcid":false,"given":"Greg","family":"Hussack","sequence":"additional","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5240-4598","authenticated-orcid":false,"given":"Jamshid","family":"Tanha","sequence":"additional","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"},{"name":"Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON K1H 8M5, Canada"},{"name":"Center for Infection, Immunity, and Inflammation (CI3), University of Ottawa, Ottawa, ON K1H 8M5, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5377-7193","authenticated-orcid":false,"given":"Bassel","family":"Akache","sequence":"additional","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tyler M.","family":"Renner","sequence":"additional","affiliation":[{"name":"Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1038\/s41587-020-0631-z","article-title":"A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction","volume":"38","author":"Tan","year":"2020","journal-title":"Nat. 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