Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Jazan Region Southwest Saudi Arabia

Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Jazan Region Southwest Saudi Arabia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Jazan Region Southwest Saudi Arabia Ommer M. Dafalla, Abdulah A. Alashor, Mohammed O. Hussien, Elsiddig M. Noureldin, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7886019/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus with high public health and veterinary importance in Africa and the Middle East. Serological surveillance, functional neutralization testing, and virus titration via tissue culture are important for monitoring the effectiveness of vaccines and outbreaks. Objective This study aimed to increase the precision of immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing. Methods Twenty serum samples were tested using the ID.vet RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and TCID₅₀/ml was calculated using the Reed and Muench method. VNT was performed at 24, 48, 72, and 96 hours after infection with different viral doses (100 to 100,000 TCID₅₀/ml), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the control, protection was determined by CPE inhibition. Results ELISA revealed robust antibody signals up to a 1:32 dilution, with S/N < 40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration of 10⁶. ⁵ TCID₅₀/ml. The VNT was time and dose- dependent, with good protection obtained at low serum dilutions and viral titers, with up to 97 protective effects at 1:2–1:8 dilutions against 100–1000 TCID₅₀/ml; however, this protection decreased at higher doses and higher serum dilutions. The results of ELISA and VNT were strongly correlated in the determination at low dilutions, whereas ELISA had decreased sensitivity at high dilutions, at which VNT was still capable of detecting neutralizing activity. Conclusion This combined strategy validates that competitive ELISA is applicable for early and medium antibody detection and VNT can functionally validate immune protection. These results provide evidence for the added value of the combination of these two methods in assessing RVFV vaccine-induced immunity and contribute to the further interpretation of antibody–virus kinetics. Biological sciences/Biological techniques Biological sciences/Biotechnology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Rift Valley fever virus Smithburn vaccine Virus neutralization test Competitive ELISA TCID₅₀ Antibody response Vero cells CPE Immunodiagnostic Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7886019","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":540648072,"identity":"b97fe0b1-aad1-4694-8c6e-e5fa0368b337","order_by":0,"name":"Ommer M. 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Arabia","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rift Valley fever virus, Smithburn vaccine, Virus neutralization test, Competitive ELISA, TCID₅₀, Antibody response, Vero cells, CPE, Immunodiagnostic","lastPublishedDoi":"10.21203/rs.3.rs-7886019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7886019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eRift Valley fever virus (RVFV) is a mosquito-borne zoonotic virus\u0026ensp;with high public health and veterinary importance in Africa and the Middle East. Serological surveillance, functional neutralization testing, and virus titration via tissue culture are important for monitoring the effectiveness of vaccines and outbreaks.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aimed to increase the precision of\u0026ensp;immunological assessments after RVFV vaccination and provide a methodological approach to combine viral quantification, serological detection and functional neutralization testing.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTwenty serum samples were tested using the ID.vet RVFV competitive ELISA to detect antibodies specific to the viral nucleocapsid protein. Viral titration was conducted in Vero cells, and TCID₅₀/ml was calculated using the Reed and Muench\u0026ensp; method. VNT was performed at 24, 48, 72, and 96 hours after infection with different viral doses (100 to 100,000\u0026ensp;TCID₅₀/ml), and the neutralizing ability of serial serum dilutions (1:2 to 1:1024) was tested. Compared with the \u0026ensp;control, protection was determined by CPE inhibition.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eELISA revealed robust antibody signals up to a 1:32 dilution, with S/N\u0026thinsp;\u0026lt;\u0026thinsp;40%, whereas for higher dilutions, antibody detection became inconclusive or negative. Virus titration was performed to verify a stock concentration\u0026ensp; of 10⁶. ⁵ TCID₅₀/ml. The VNT was time and dose- dependent, with good\u0026ensp; protection obtained at low serum dilutions and viral titers, with up to 97 protective effects at 1:2\u0026ndash;1:8 dilutions against 100\u0026ndash;1000 TCID₅₀/ml; however, this protection decreased at higher doses and higher serum dilutions. The results of ELISA and VNT were strongly correlated in the determination at low dilutions, whereas ELISA had decreased sensitivity at high dilutions, at which VNT was still capable\u0026ensp;of detecting neutralizing activity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis combined strategy validates that competitive ELISA\u0026ensp; is applicable for early and medium antibody detection and VNT can functionally validate immune protection. These results provide evidence for the added value of the combination of these two methods in assessing RVFV vaccine-induced immunity and contribute to the\u0026ensp; further interpretation of antibody\u0026ndash;virus kinetics.\u003c/p\u003e","manuscriptTitle":"Integrated Assessment of Antibody Responses to RVFV Using Competitive ELISA and VNT in Vaccinated Animal Samples from Jazan Region Southwest Saudi Arabia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 11:11:54","doi":"10.21203/rs.3.rs-7886019/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62fed124-a094-4530-be05-21cf6509c176","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57514252,"name":"Biological sciences/Biological techniques"},{"id":57514253,"name":"Biological sciences/Biotechnology"},{"id":57514254,"name":"Health sciences/Diseases"},{"id":57514255,"name":"Biological sciences/Immunology"},{"id":57514256,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-07T08:40:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 11:11:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7886019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7886019","identity":"rs-7886019","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}