Single-Chain Variable Fragment-Based Dot Blot, Single, and Multiple Assays for Rapid SARS-CoV-2 Diagnostics

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dc.contributor.authorCam Derin, Dilek
dc.contributor.authorGultekin, Enes
dc.contributor.authorTaskin, Irmak Icen
dc.contributor.authorDundar, Muhammed
dc.contributor.authorOtlu, Baris
dc.date.accessioned2026-04-04T13:33:31Z
dc.date.available2026-04-04T13:33:31Z
dc.date.issued2025
dc.departmentİnönü Üniversitesi
dc.description.abstractBackground Antigenic detection is reliably utilized in rapid diagnostic tests and provides a significant time advantage during pandemics and epidemics. Therefore, the rapid detection of viral infections is of great importance and will remain crucial in the future. The SARS-CoV-2 outbreak, which resulted in severe losses, is the most recent example of this necessity. Among rapid diagnostic tests, lateral flow assays (LFAs) are the most practical and do not require specialized equipment, typically being developed using antibody pairs.Objective This study aimed to recombinantly produce a single-chain variable fragment (scFv) specific to the SARS-CoV-2 spike receptor-binding domain (sRBD) and to employ it in the development of LFAs utilizing both antibody and aptamer pairs and an aptamer cocktail.Methods Gold nanoparticles were employed as labeling agents, while both the scFv and full length forms of CR3022, along with aptamers specific to the S and N proteins, were utilized in a sandwich assay format.Results scFv was produced at a higher concentration and biologically active. It demonstrated effective viral detection in single LFA, dot blot assay (DBA), and multiplex LFA. While single LFA successfully detected only the synthetic target, DBA and multiplex LFA selectively identified the virus in nasopharyngeal and oropharyngeal swab samples.Conclusion Findings highlight the differences and effectiveness of using scFv in combination with other capture agents and different assay principles for the development of cost-effective and rapid diagnostic tests.Highlights scFvs exhibit variable binding in sandwich assays depending on the combinations employed. When used in combination with an aptamer cocktail, scFvs demonstrate enhanced target binding, which is shown for the first time in this study. The use of multiple testing strategies enables a more effective viral diagnosis.
dc.description.sponsorshipScientific and Technological Research Council of Turkey
dc.description.sponsorshipThis work was supported by the Scientific and Technological Research Council of Turkey (TUB & Idot;TAK; 120Z962).
dc.identifier.doi10.1093/jaoacint/qsaf105
dc.identifier.issn1060-3271
dc.identifier.issn1944-7922
dc.identifier.orcid0000-0001-6509-2012
dc.identifier.orcid0000-0001-5617-4067
dc.identifier.orcid0000-0002-1612-0563
dc.identifier.orcid0000-0002-8545-1868
dc.identifier.orcid0000-0002-6220-0521
dc.identifier.pmid41352973
dc.identifier.urihttps://doi.org/10.1093/jaoacint/qsaf105
dc.identifier.urihttps://hdl.handle.net/11616/109183
dc.identifier.wosWOS:001649122100001
dc.identifier.wosqualityQ3
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakPubMed
dc.language.isoen
dc.publisherOxford Univ Press Inc
dc.relation.ispartofJournal of Aoac International
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanı
dc.rightsinfo:eu-repo/semantics/openAccess
dc.snmzKA_WOS_20250329
dc.subjectInstrument-Free
dc.subjectSpike Protein
dc.subjectDna Aptamer
dc.subjectLow-Cost
dc.subjectAntibody
dc.subjectScfv
dc.subjectDiscovery
dc.subjectBiosensor
dc.subjectDomain
dc.titleSingle-Chain Variable Fragment-Based Dot Blot, Single, and Multiple Assays for Rapid SARS-CoV-2 Diagnostics
dc.typeArticle

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