{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T10:10:40Z","timestamp":1789639840461,"version":"4.0.0"},"reference-count":38,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T00:00:00Z","timestamp":1773792000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>\n                    Neutralizing antibody (nAb) assays are essential for evaluating antibody-mediated immune response against SARS-CoV-2, in the context of efficacy and potency of vaccines, as well as population-level surveillance. This study aimed to estimate the associated uncertainty during the validation of two neutralization assays\u201450% tissue culture infectious dose microneutralization (TCID\n                    <jats:sub>50<\/jats:sub>\n                    MN) and fluorescent focus reduction neutralization test (FRNT)\u2014to measure activity of nAbs against SARS-CoV-2 variants of concern (VOCs) within an ISO\/IEC 17025:2017 accredited laboratory. Human serum samples were tested against five SARS-CoV-2 variants (Wuhan, BA.2, BA.5, XBB.1, and JN.1) using vesicular stomatitis virus (VSV) pseudotyped viruses. The validation process was based on international guidelines for the suitability and validation of analytical methods. Both assays demonstrated high diagnostic performance to detect neutralizing activity, where FRNT yielded higher sensitivity but greater inter-analyst variability than TCID\n                    <jats:sub>50<\/jats:sub>\n                    MN. These results highlight the importance of identifying the level of impact of factors associated with obtaining a result, such as the training of analysts and the standardization of the test. This study establishes a validated framework for nAb quantification in compliance with international standards. The declaration of associated uncertainty to each method, along with sustained internal quality control, strengthens the diagnostic capacity of these assays in clinical research of correlates of protection and immunity induced by vaccination, in addition to reinforcing transparency and regulatory compliance, as critical elements in decision-making.\n                  <\/jats:p>","DOI":"10.3389\/fimmu.2026.1768395","type":"journal-article","created":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T06:56:21Z","timestamp":1773816981000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Associated uncertainty estimation during the validation process of TCID50 and FRNT neutralization assays against SARS-CoV-2 variants, used in population surveillance research and correlates of protection"],"prefix":"10.3389","volume":"17","author":[{"given":"Edgar","family":"Reyna-Rosas","sequence":"first","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milton","family":"Nieto-Ponce","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Palencia-Reyes","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Blancas-Ru\u00edz","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Carranza","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martha","family":"Torres","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Horacio","family":"Zamudio-Meza","sequence":"additional","affiliation":[{"name":"Laboratorio de Inmunobiolog\u00eda de la Tuberculosis, Instituto Nacional de Enfermedades Respiratorias Ismael Cos\u00edo Villegas","place":["Mexico City, Mexico"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,3,18]]},"reference":[{"key":"B1","volume-title":"StatPearls [Internet]","author":"Cascella","year":"2024"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1038\/s41564-020-0688-y","article-title":"Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses","volume":"5","author":"Letko","year":"2020","journal-title":"Nat Microbiol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1038\/s41418-020-00720-9","article-title":"COVID-19 vaccines: where we stand and challenges ahead","volume":"28","author":"Forni","year":"2021","journal-title":"Cell Death Differ"},{"key":"B4","unstructured":"Vacunas autorizadas COVID-19\n          \n          \n          2025"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.smim.2020.101413","article-title":"Transforming vaccine development","volume":"50","author":"Black","year":"2020","journal-title":"Semin Immunol"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1002\/rmv.2313","article-title":"Covid-19 vaccines and variants of concern: A review","volume":"32","author":"Hadj Hassine","year":"2022","journal-title":"Rev Med Virol"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3390\/v14071560","article-title":"Overview of neutralization assays and international standard for detecting SARS-CoV-2 neutralizing antibody","volume":"14","author":"Liu","year":"2022","journal-title":"Viruses"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines12050554","article-title":"SARS-CoV-2 neutralization assays used in clinical trials: A narrative review","volume":"12","author":"Sun","year":"2024","journal-title":"Vaccines"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-3618-2_25","article-title":"Neutralization assay for chikungunya virus infection: Plaque reduction neutralization test","volume":"1426","author":"Azami","year":"2016","journal-title":"Methods Mol Biol (Clifton NJ)"},{"key":"B10","volume-title":"Medical microbiology","author":"Albrecht","year":"1996"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.3390\/diseases12010029","article-title":"Plaque reduction neutralization test (PRNT) accuracy in evaluating humoral immune response to SARS-CoV-2","volume":"12","author":"Horbach","year":"2024","journal-title":"Dis (Basel Switzerland)"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-021-00536-y","article-title":"Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaque reduction neutralization, microneutralization and pseudotyped virus neutralization assays","volume":"16","author":"Bewley","year":"2021","journal-title":"Nat Protoc"},{"key":"B13","doi-asserted-by":"publisher","first-page":"475","DOI":"10.1016\/j.chom.2020.06.021","article-title":"Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2","volume":"28","author":"Case","year":"2020","journal-title":"Cell Host Microbe"},{"key":"B14","article-title":"Manual for the virological investigation of poliomyelitis","year":"1990"},{"key":"B15","article-title":"Guideline on clinical evaluation of vaccines (EMEA\/CHMP\/VWP\/164653\/05 Rev. 1)","year":"2023"},{"key":"B16","article-title":"ICH Q2(R2) Guideline on validation of analytical procedures (EMA\/CHMP\/ICH\/82072\/2006)","year":"2023"},{"key":"B17","article-title":"ISO 9000:2015(es). Sistemas de gesti\u00f3n de la calidad Fundamentos y vocabulario","year":"2015"},{"key":"B18","article-title":"ISO\/IEC 17025:2017(es). Requisitos generales para la competencia de los laboratorios de ensayo y calibraci\u00f3n. Geneva: International Organization for Standardization","year":"2017"},{"key":"B19","volume-title":"Eurachem Guide: The fitness for purpose of analytical methods \u2013 A laboratory guide to method validation and related topics","author":"Magnusson","year":"2014"},{"key":"B20","article-title":"ICH M4Q(R2) Guideline on the Common Technical Document for the registration of pharmaceuticals for human use: Quality (EMA\/CHMP\/ICH\/178042\/2025)","year":"2025"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2024.126511","article-title":"Phase II study on the safety and immunogenicity of single-dose intramuscular or intranasal administration of the AVX\/COVID-12 \"Patria\" recombinant Newcastle disease virus vaccine as a heterologous booster against COVID-19 in Mexico","volume":"43","author":"L\u00f3pez-Mac\u00edas","year":"2025","journal-title":"Vaccine"},{"key":"B22","article-title":"Evaluaci\u00f3n de datos de medici\u00f3n. Gu\u00eda para la expresi\u00f3n de la incertidumbre de medida. Edici\u00f3n digital","year":"2008"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.5501\/wjv.v5.i2.85","article-title":"Determination of 50% endpoint titer using a simple formula","volume":"5","author":"Ramakrishnan","year":"2016","journal-title":"World J Virol"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1177\/20499361251314763","article-title":"The JN.1 variant of COVID-19: immune evasion, transmissibility, and implications for global health","volume":"12","author":"Naveed Siddiqui","year":"2025","journal-title":"Ther Adv Infect Dis"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1002\/bit.28703","article-title":"Vaccine process technology-a decade of progress","volume":"121","author":"Buckland","year":"2024","journal-title":"Biotechnol Bioeng"},{"key":"B26","doi-asserted-by":"publisher","first-page":"34","DOI":"10.1208\/s12248-022-00685-2","article-title":"Analytical quality by design, life cycle management, and method control","volume":"24","author":"Verch","year":"2022","journal-title":"AAPS J"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.02020","article-title":"Evaluation of neutralizing antibodies against highly pathogenic coronaviruses: A detailed protocol for a rapid evaluation of neutralizing antibodies using vesicular stomatitis virus pseudovirus-based assay","volume":"11","author":"Almahboub","year":"2020","journal-title":"Front Microbiol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-020-0394-5","article-title":"Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay","volume":"15","author":"Nie","year":"2020","journal-title":"Nat Protoc"},{"key":"B29","doi-asserted-by":"publisher","DOI":"10.1002\/cpim.116","article-title":"Development of a rapid focus reduction neutralization test assay for measuring SARS-CoV-2 neutralizing antibodies","volume":"131","author":"Vanderheiden","year":"2020","journal-title":"Curr Protoc Immunol"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2021.05.063","article-title":"Evidence for antibody as a protective correlate for COVID-19 vaccines","volume":"39","author":"Earle","year":"2021","journal-title":"Vaccine"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-021-01377-8","article-title":"Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection","volume":"27","author":"Khoury","year":"2021","journal-title":"Nat Med"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3201\/eid2902.221422","article-title":"Correlates of protection, thresholds of protection, and immunobridging among persons with SARS-CoV-2 infection","volume":"29","author":"Khoury","year":"2023","journal-title":"Emerg Infect Dis"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2024.e34925","article-title":"Validation of a focus reduction neutralization test (FRNT) to rapidly titrate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 universal analyzer","volume":"10","author":"Puglia","year":"2024","journal-title":"Heliyon"},{"key":"B34","doi-asserted-by":"publisher","first-page":"4059","DOI":"10.1038\/s41467-020-17892-0","article-title":"A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation","volume":"11","author":"Muruato","year":"2020","journal-title":"Nat Commun"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2024.114945","article-title":"Validation of a severe acute respiratory syndrome coronavirus 2 microneutralization assay for evaluation of vaccine immunogenicity","volume":"327","author":"Hamilton","year":"2024","journal-title":"J Virol Methods"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.3390\/vaccines12040374","article-title":"Development and validation of a plaque assay to determine the titer of a recombinant live-attenuated viral vaccine for SARS-CoV-2","volume":"12","author":"Toister","year":"2024","journal-title":"Vaccines (Basel)"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.03789-22","article-title":"Validation and establishment of the SARS-CoV-2 lentivirus surrogate neutralization assay as a prescreening tool for the plaque reduction neutralization test","volume":"11","author":"Merluza","year":"2023","journal-title":"Microbiol Spectr"},{"key":"B38","doi-asserted-by":"publisher","first-page":"23921","DOI":"10.1038\/s41598-021-03154-6","article-title":"Calibration of two validated SARS-CoV-2 pseudovirus neutralization assays for COVID-19 vaccine evaluation","volume":"11","author":"Huang","year":"2021","journal-title":"Sci Rep"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1768395\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T06:56:25Z","timestamp":1773816985000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2026.1768395\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,18]]},"references-count":38,"alternative-id":["10.3389\/fimmu.2026.1768395"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2026.1768395","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,18]]},"article-number":"1768395"}}