Challenges and Methodological Aspects of Detecting Anti-RBD Antibodies by ELISA in SARS-CoV-2 Research

Challenges and Methodological Aspects of Detecting Anti-RBD Antibodies by ELISA in SARS-CoV-2 Research | 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 Data Note Challenges and Methodological Aspects of Detecting Anti-RBD Antibodies by ELISA in SARS-CoV-2 Research Giovanna Santos Oliveira, Amanda Izeli Portilho, Valéria Oliveira Silva, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9031844/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 The detection of IgE antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 has been associated with aspects of COVID-19 severity, including potential mast cell–mediated mechanisms. Recent studies analyzed IgE anti-RBD across different COVID-19 severity groups using ELISA. The coating-antigen concentration influences the detection capacity of the immunoassay; thus, low-abundance antibodies, such as IgE, may require higher antigen concentrations to achieve optimal experimental conditions. Here, we present the effect of different coating concentrations for anti-RBD IgE and IgG4 detection in samples of vaccinated individuals. Our results suggested that intermediate antigen concentrations provide a better balance between sensitivity and specificity, highlighting the importance of methodological standardization. Immunology SARS-CoV-2 RBD IgE IgG4 indirect ELISA assay sensitivity Figures Figure 1 Background The recent study of De la Poza et al. (2025), published in the Virology Journal, investigated the presence of IgE antibodies directed against the Receptor binding domain (RBD) of SARS-CoV-2 in patients with different clinical presentations of COVID-19, according to disease severity, proposing an association between IgE responses, mast-cell activation, and the hyperinflammatory response observed in later stages of the disease. The identification of IgE antibodies in this context represents an important contribution, as antibody-mediated mechanisms extend beyond viral neutralization and involve complex immunoregulatory functions [1]. Antibodies exert multiple biological functions, including pathogen neutralization, complement activation, and promotion of phagocytosis, with these effects being modulated by antibody class, subclass, and interactions with Fc receptors [2,3]. Importantly, different immunoglobulin isotypes circulate in serum at markedly distinct concentrations. While IgG and IgM are present at high levels, being detectable even at limited antigen coating concentrations in immunoassays, low-abundance immunoglobulins such as IgE and IgG4 are present at much lower concentrations, rendering their detection particularly sensitive to experimental conditions in immunoenzymatic assays [4,5]. Previous studies have demonstrated that the magnitude and profile of humoral immune responses to SARS-CoV-2 vary according to disease severity, including differences in antibody kinetics and intensity, further emphasizing the need for sensitive and carefully optimized methodologies when assessing specific antibody classes and subclasses [6]. Critical assessment and methodological considerations One methodological aspect of the study by de la Poza et al. that warrants discussion is the antigen concentration used for IgE detection by ELISA. In that work, recombinant SARS-CoV-2 RBD was employed at a coating concentration of 10 µg/mL . High antigen density may increase binding capacity, but assays targeting low-abundance immunoglobulins are particularly susceptible to changes in signal-to-noise ratio and to increased nonspecific binding under conditions of antigen excess. The use of lower RBD concentrations for IgE and IgG4 detection has been described for the first time in studies conducted by our group. Previous work standardized coating concentration of 1 µg/mL of recombinant RBD, enabling sensitive detection of antigen-specific IgE and IgG4 with reduced nonspecific background signals [7]. The parallel evaluation of the IgG4 subclass was used as a complementary methodological reference for low-abundance immunoglobulins. Widely adopted SARS-CoV-2 ELISA protocols were standardized before employing different RBD concentrations for different isotypes. While our investigation of IgE and IgG4 used 1 µg/mL [7], Stadlbauer et al. (2020) used 2 µg/mL in a broadly validated serological screening protocol for IgG [8]. Similarly, Moura et al. (2021) and Costa et al. (2024) employed 2.5 µg/mL of RBD for the detection of IgG and its subclasses, achieving appropriate analytical performance [5,9]. Together, these examples indicate that intermediate RBD concentrations are often sufficient, underscoring the need for standardized antigen-coating conditions for each antibody class. In addition, differences in ELISA plate surface chemistry and binding capacity may significantly influence antigen immobilization and antibody detection, thereby affecting assay sensitivity and specificity [10]. Exploratory evidence and practical implications In addition to the conceptual considerations described above, comparative experimental analyses were performed to assess the impact of antigen density on the detection of IgE and IgG4. 12 individuals, known to have anti-RBD IgE, were selected within a larger COVID-19 cohort (CAAE 31924420.8.0000.0059). Each individual provided 3 samples: before the vaccine, after two and after three vaccine doses. 6 of those were vaccinated with a CoronaVac (Instituto Butantan)+Comirnaty (Pfizer-BioNTech) schedule, and the other 6 received a Vaxzevria (AstraZeneca)+Comirnaty schedule. 12 pre-pandemic samples were used as controls [7]. Briefly, high-binding plates were coated with different RBD concentrations: 1, 2, 5, and 10 µg/mL. Sera samples were incubated at a 1:5 dilution, followed by anti-IgE-ε chain or anti-IgG4 (Sigma-Aldrich) at a 1:1000 dilution, and the reactions were revealed with TMB substrate (Sigma-Aldrich). More details about the cohort, RBD production and ELISA protocols are described in Costa et al. (2024) and Portilho et al. (2024). Within this exploratory context, increasing RBD coating concentrations did not affect reactivity in pre-pandemic control samples. For IgE, antibody reactivity remained low in pre-vaccination samples and increased after the second and, more markedly, after the third vaccine dose, with a clear improvement in signal observed when moving from lower coating concentrations (1–2 µg/mL) to an intermediate concentration. Notably, IgE reactivity showed minimal differences between 5 and 10 µg/mL, supporting the selection of 5 µg/mL as an optimal coating concentration that balances assay performance and reagent consumption (Figure 1A–D). A similar pattern was observed for IgG4, a low-abundance serum immunoglobulin that is generally present at higher concentrations than IgE, with minimal reactivity before vaccination and a modest increase predominantly after the third dose, while control samples remained unaffected regardless of the antigen concentration used (Figure 1E–H). Noteworthy, anti-SARS-CoV-2 IgG4 is mostly induced after mRNA vaccination schedules or when multiple booster doses are administered [11,12]. Finally, although no increase in background was observed in this analysis, optimal antigen concentrations and relative assay sensitivity may vary across laboratories depending on protocol design, reagents, and cohort characteristics. Beyond antigen concentration, the physicochemical nature of the antigen itself represents a critical factor in defining immunoenzymatic assay conditions, influencing adsorption to the solid phase and epitope accessibility. These variables directly affect assay reliability and reproducibility, particularly when evaluating immunoglobulins present at low concentrations. Conclusion The study by de la Poza et al. provides valuable insights into IgE responses associated with COVID-19. However, given the intrinsic sensitivity of these assays to experimental conditions, careful selection of antigen concentration and coating parameters is essential for robust data interpretation. Regarding the role of low immunoglobulin levels in the immunopathogenesis of COVID-19, the adoption of standardized and empirically validated conditions, including the use of 5 µg/mL RBD concentrations for IgE and IgG4, may improve assay reproducibility and evidence-based conclusions. Abbreviations Ig - Immunoglobulin RBD - Receptor-binding domain SARS-CoV-2 - Severe acute respiratory syndrome coronavirus 2 COVID-19 - Coronavirus disease 2019 ELISA - Enzyme-linked immunosorbent assay Declarations Ethics approval and consent to participate This study was registered and approved by the institutional ethics committee of Adolfo Lutz Institute (CAAE 31924420.8.0000.0059), and written informed consent was provided by all participants. Additionally, all experiments were performed following relevant guidelines and regulations. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP (grants numbers 2018/04202-0, 2018/14384-9, 2021/11936-3, 2022/05566-1); Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq (grants numbers 305301/2022-5, MS-DIAHV 24/2019 process 442776/2019-5, 132059/2025-8); Financiadora de Estudos e Projetos (Grant Number 01160075); Fundo de Investimento para Educação Sanitária e Imunização em Massa contra Doenças Transmissíveis/FESIMA (Grants Numbers 011/2021, 59/2021) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/CAPES (finance code 001). Authors' contributions Conceptualization and study design were performed by GSO, AIP, and EDG. Volunteer recruitment and data collection were performed by VOS and LFMB. Antigen production was carried out by HHMC and CRP. Laboratory experiments were performed by GSO, AIP, AFS, NSM, and EDG. Sample management was conducted by VOS and LFMB. Statistical analysis was performed by GSO and AIP. The manuscript was written by GSO, AIP, AFS, and NSM, and EDG revised the original draft. All authors contributed to the study, commented on previous versions of the manuscript, and approved the final manuscript. Acknowledgements The authors would like to thank Dr Florian Krammer (Mount Sinai Hospital, New York, NY, USA) for donating the plasmid to produce the RBD to Dr Carlos Roberto Prudencio. References de la Poza, J.F.D., Parés, A.R., Aparicio-Calvente, I. et al. Frequency of IgE antibody response to SARS-CoV-2 RBD protein across different disease severity COVID19 groups. Virol J 22, 58 (2025). doi: 10.1186/s12985-025-02677-y. Heyman B. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu Rev Immunol. 2000;18:709-737. doi:10.1146/annurev.immunol.18.1.709. Daëron M. The function of antibodies. Immunol Rev. 2024 Nov;328(1):113-125. doi: 10.1111/imr.13387. Khan SR, Chaker L, Ikram MA, Peeters RP, van Hagen PM, Dalm VASH. Determinants and Reference Ranges of Serum Immunoglobulins in Middle-Aged and Elderly Individuals: a Population-Based Study. J Clin Immunol. 2021 Nov;41(8):1902-1914. doi: 10.1007/s10875-021-01120-5. Moura, AD, da Costa, HHM, Correa, VA, Lima, AKS, Lindoso, JAL, Gaspari, ED, et al. Serological assessment of SARS-CoV-2 infection and vaccination using ELISA-based assays. Sci Rep 11 , 17642 (2021). doi: 10.1038/s41598-021-95045-z. Plūme J, Galvanovskis A, Šmite S, Romanchikova N, Zayakin P, Linē A. Early and strong antibody responses to SARS-CoV-2 predict disease severity in COVID-19 patients. J Transl Med. 2022 Apr 15;20(1):176. doi: 10.1186/s12967-022-03382-y. Portilho AI, Silva VO, Da Costa HHM, Yamashiro R, de Oliveira IP, de Campos IB, et al. An unexpected IgE anti–receptor-binding domain response following natural infection and different types of SARS-CoV-2 vaccine. Sci Rep. 2024 Aug 28;14(1):20003. doi: 10.1038/s41598-024-71047-5. Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, et al. SARS-CoV-2 seroconversion in humans: a detailed protocol for a serological assay. Curr Protoc Microbiol. 2020 Jun;57(1):e100. Doi: 10.1002/cpmc.100. da Costa HHM, Silva VO, Amorim GC, Guereschi MG, Sergio LM, Gomes CHR, et al. Assessment of an in-house IgG ELISA targeting SARS-CoV-2 RBD for detection of antibody responses. J Immunol Methods. 2024 Jul;530:113683. doi: 10.1016/j.jim.2024.113683. Crowther JR. The ELISA guidebook. 2nd ed. Methods in Molecular Biology. Totowa (NJ): Humana Press; 2009. doi:10.1007/978-1-60327-254-4. Kiszel P, Sík P, Miklós J, Kajdácsi E, Sinkovits G, Cervenak L, et al. Class switch towards spike protein-specific IgG4 antibodies after SARS-CoV-2 mRNA vaccination depends on prior infection history. Sci Rep. 2023 Aug 13;13(1):13166. doi: 10.1038/s41598-023-40103-x. Irrgang P, Gerling J, Kocher K, Lapuente D, Steininger P, Habenicht K, et al. Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination. Sci Immunol. 2023 Jan 27;8(79):eade2798. doi: 10.1126/sciimmunol.ade2798. Additional Declarations The authors declare no competing interests. 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. 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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-9031844","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Data Note","associatedPublications":[],"authors":[{"id":600728250,"identity":"29214531-ed54-4e43-b635-618a3f7f3e48","order_by":0,"name":"Giovanna Santos Oliveira","email":"","orcid":"https://orcid.org/0009-0003-2641-0323","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Giovanna","middleName":"Santos","lastName":"Oliveira","suffix":""},{"id":600728251,"identity":"a569391c-b674-46e4-9e7e-2a080bd9a054","order_by":1,"name":"Amanda Izeli Portilho","email":"","orcid":"https://orcid.org/0000-0003-0875-5910","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"Izeli","lastName":"Portilho","suffix":""},{"id":600728252,"identity":"0f916014-3f4c-4980-965e-907049debda1","order_by":2,"name":"Valéria Oliveira Silva","email":"","orcid":"https://orcid.org/0000-0002-5346-9633","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Valéria","middleName":"Oliveira","lastName":"Silva","suffix":""},{"id":600728253,"identity":"db84221d-0464-478e-bcde-a3dc4ef8630a","order_by":3,"name":"Ana Flavia Segati","email":"","orcid":"https://orcid.org/0000-0002-9087-7898","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Flavia","lastName":"Segati","suffix":""},{"id":600728254,"identity":"2e94db1c-8326-459e-a8a4-d9fc8c77e261","order_by":4,"name":"Nicoly Simões de Melo","email":"","orcid":"https://orcid.org/0000-0003-4538-1399","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Nicoly","middleName":"Simões","lastName":"de Melo","suffix":""},{"id":600728256,"identity":"dced17bd-ea82-4940-86b4-23c0db8d5d40","order_by":5,"name":"Hernan Hermes Monteiro da Costa","email":"","orcid":"https://orcid.org/0000-0001-8972-749X","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Hernan","middleName":"Hermes Monteiro da","lastName":"Costa","suffix":""},{"id":600728257,"identity":"070d1e8d-c869-48f1-b7b0-c2ca8893db1d","order_by":6,"name":"Carlos Roberto Prudencio","email":"","orcid":"https://orcid.org/0000-0001-7701-0707","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Roberto","lastName":"Prudencio","suffix":""},{"id":600728258,"identity":"5b2bf32a-4c31-44cf-b46a-8a4459d57c92","order_by":7,"name":"Luís Fernando de Macedo Brígido","email":"","orcid":"https://orcid.org/0000-0002-1022-7837","institution":"Adolfo Lutz Institute","correspondingAuthor":false,"prefix":"","firstName":"Luís","middleName":"Fernando de Macedo","lastName":"Brígido","suffix":""},{"id":600728259,"identity":"4905adc9-0419-43d5-9675-b7bd24c99417","order_by":8,"name":"Elizabeth De Gaspari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABQElEQVRIie3RsUrDQBjA8e84uCyJt14I4it8xUGLUl/lQqGjDlkEoV4JXBZ9Cl/AMWPKQbvc4qaLBAJuQruZQfRKKbVp7SyYPyFwl/vxBQ6gre0v5hEF7gFcbTBvpKBcrRafmtElEWvijxXIvWS5vyYg3Pl9hFOazur8fHgC3tO8zntXB2E1mkkYXgJ/mAiSvzZJmBLVubcD0VV+EgW239VRnLpBJgHxNhDEJk2ChigZaCOw8GVENEUWxcqRwr3tMRItm+TCkfGn/lqQfl3rW2ThOP1wP/YrQUpGaaALR7yJcOOQCaLdFBorftcpdxBhSEoPbT98ND47C+wUmR/rU4kmYYLFZWy3CM+yav6e9zhOs+qlzm/wKJtWz7Pr4SXnpijm+RZZR338cQsIkm1c0M68cuPiJPBi7/m2tra2f9M3Di5uyP+29rQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8332-2248","institution":"Adolfo Lutz Institute","correspondingAuthor":true,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"De Gaspari","suffix":""}],"badges":[],"createdAt":"2026-03-04 15:22:36","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9031844/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9031844/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103986387,"identity":"09f632ca-d676-4e94-8221-e83bf173a21a","added_by":"auto","created_at":"2026-03-05 10:37:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4491546,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of RBD coating concentration on IgE and IgG4 detection by ELISA on high-binding plates. Exploratory analysis of anti–SARS-CoV-2 RBD IgE and IgG4 reactivity assessed across vaccination stages. Recombinant RBD was immobilized at 1, 2, 5, or 10 μg/mL, and antibody levels are shown as optical density at 450 nm (OD₄₅₀). (A–D) IgE reactivity measured at 1 μg/mL (A), 2 μg/mL (B), 5 μg/mL (C), and 10 μg/mL (D). (E–H) IgG4 reactivity measured at 1 μg/mL (E), 2 μg/mL (F), 5 μg/mL (G), and 10 μg/mL (H). Statistical comparisons among vaccination stages were performed using the Friedman test followed by Dunn’s multiple comparisons post hoc test. Asterisks indicate levels of statistical significance (* p \u0026lt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.001); ns - not significant.\u003c/p\u003e","description":"","filename":"IgExIgG4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9031844/v1/79232899834a8844e8c0ff6e.jpg"},{"id":104401746,"identity":"a263d83c-7a9f-4ce7-b1e2-ac73977a9b37","added_by":"auto","created_at":"2026-03-11 12:13:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4928998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9031844/v1/bde8e36a-1224-48da-a957-fe7b1857e272.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eChallenges and Methodological Aspects of Detecting Anti-RBD Antibodies by ELISA in SARS-CoV-2 Research\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThe recent study of De la Poza et al. (2025), published in the Virology Journal,\u0026nbsp; investigated the presence of IgE antibodies directed against the Receptor binding domain (RBD) of SARS-CoV-2 in patients with different\u0026nbsp;clinical presentations of COVID-19, according to disease severity, proposing an association between IgE responses, mast-cell activation, and the hyperinflammatory response observed in later stages of the disease. The identification of IgE antibodies in this context represents an important contribution, as antibody-mediated mechanisms extend beyond viral neutralization and involve complex immunoregulatory functions [1].\u003c/p\u003e\n\u003cp\u003eAntibodies exert multiple biological functions, including pathogen neutralization, complement activation, and promotion of phagocytosis, with these effects being modulated by antibody class, subclass, and interactions with Fc receptors [2,3].\u0026nbsp;Importantly, different immunoglobulin isotypes circulate in serum at markedly distinct concentrations. While IgG and IgM are present at high levels, being detectable even at limited antigen coating concentrations in immunoassays, low-abundance immunoglobulins such as IgE and IgG4 are present at much lower concentrations, rendering their detection particularly sensitive to experimental conditions in immunoenzymatic assays [4,5].\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that the magnitude and profile of humoral immune responses to SARS-CoV-2 vary according to disease severity, including differences in antibody kinetics and intensity, further emphasizing the need for sensitive and carefully optimized methodologies when assessing specific antibody classes and subclasses [6].\u003c/p\u003e"},{"header":"Critical assessment and methodological considerations","content":"\u003cp\u003eOne methodological aspect of the study by de la Poza et al. that warrants discussion is the antigen concentration used for IgE detection by ELISA. In that work, recombinant SARS-CoV-2 RBD was employed at a coating concentration of 10 µg/mL\u003cstrong\u003e.\u003c/strong\u003e High antigen density may increase binding capacity, but assays targeting low-abundance immunoglobulins are particularly susceptible to changes in signal-to-noise ratio and to increased nonspecific binding under conditions of antigen excess.\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe use of lower RBD concentrations for IgE and IgG4 detection has been described for the first time in studies conducted by our group. Previous work standardized coating concentration of 1 µg/mL of recombinant RBD, enabling sensitive detection of antigen-specific IgE and IgG4 \u0026nbsp;with reduced nonspecific background signals [7]. The parallel evaluation of the IgG4 subclass was used as a complementary methodological reference for low-abundance immunoglobulins.\u003c/p\u003e\u003cp\u003eWidely adopted SARS-CoV-2 ELISA protocols were\u0026nbsp;standardized before employing\u0026nbsp;different RBD concentrations for different isotypes. While our investigation of IgE and IgG4 used 1 µg/mL [7], Stadlbauer et al. (2020) used 2 µg/mL in a broadly validated serological screening protocol for IgG [8]. Similarly, Moura et al. (2021) and Costa et al. (2024) employed 2.5 µg/mL of RBD\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor the detection of IgG and its subclasses, achieving appropriate analytical performance [5,9]. Together, these examples indicate that intermediate RBD concentrations are often sufficient, underscoring the need for standardized antigen-coating conditions for\u0026nbsp;each antibody class. In addition, differences in ELISA plate surface chemistry and binding capacity may significantly influence antigen immobilization and antibody detection, thereby affecting assay sensitivity and specificity [10].\u003c/p\u003e"},{"header":"Exploratory evidence and practical implications","content":"\u003cp\u003eIn addition to the conceptual considerations described above, comparative experimental analyses were performed to assess the impact of antigen density on the detection of IgE and IgG4.\u0026nbsp;12 individuals, known to have anti-RBD IgE, were selected within a larger COVID-19 cohort (CAAE 31924420.8.0000.0059). Each individual provided 3 samples: before the vaccine, after two and after three vaccine doses. 6 of those were vaccinated with a CoronaVac (Instituto Butantan)+Comirnaty (Pfizer-BioNTech) schedule, and the other 6 received a Vaxzevria (AstraZeneca)+Comirnaty schedule. 12 pre-pandemic samples were used as controls [7].\u003c/p\u003e\u003cp\u003eBriefly, high-binding plates were coated with different RBD concentrations: 1, 2, 5, and 10 µg/mL. Sera samples were incubated at a 1:5 dilution, followed by anti-IgE-ε chain or anti-IgG4 (Sigma-Aldrich) at a 1:1000 dilution, and the reactions were revealed with TMB substrate (Sigma-Aldrich). More details about the cohort, RBD production and ELISA protocols are described in Costa et al. (2024) and Portilho et al. (2024).\u0026nbsp;\u003c/p\u003e\u003cp\u003eWithin this exploratory context, increasing RBD coating concentrations did not affect reactivity in \u0026nbsp;pre-pandemic control samples. For IgE, antibody reactivity remained low in pre-vaccination samples and increased after the second and, more markedly, after the third vaccine dose, with a clear improvement in signal observed when moving from lower coating concentrations (1–2 µg/mL) to an intermediate concentration. Notably, IgE reactivity showed minimal differences between 5 and 10 µg/mL, supporting the selection of 5 µg/mL as an optimal coating concentration that balances assay performance and reagent consumption (Figure 1A–D). A similar pattern was observed for IgG4, a low-abundance serum immunoglobulin that is generally present at higher concentrations than IgE, with minimal reactivity before vaccination and a modest increase predominantly after the third dose, while control samples remained unaffected regardless of the antigen concentration used (Figure 1E–H). Noteworthy, anti-SARS-CoV-2 IgG4 is mostly induced after mRNA vaccination schedules or when multiple booster doses are administered [11,12]. Finally, although no increase in background was observed in this analysis, optimal antigen concentrations and relative assay sensitivity may vary across laboratories depending on protocol design, reagents, and cohort characteristics.\u003c/p\u003e\u003cp\u003eBeyond antigen concentration, the physicochemical nature of the antigen itself represents a critical factor in defining immunoenzymatic assay conditions, influencing adsorption to the solid phase and epitope accessibility. These variables directly affect assay reliability and reproducibility, particularly when evaluating immunoglobulins present at low concentrations.\u003c/p\u003e"},{"header":"Conclusion ","content":"\u003cp\u003eThe study by de la Poza et al. provides valuable insights into IgE responses associated with COVID-19. However, given the intrinsic sensitivity of these assays to experimental conditions, careful selection of antigen concentration and coating parameters is essential for robust data interpretation. Regarding the role of low immunoglobulin levels in the immunopathogenesis of COVID-19, the adoption of standardized and empirically validated conditions, including the use of 5 \u0026nbsp;µg/mL RBD concentrations for IgE and IgG4, may improve assay reproducibility and evidence-based conclusions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIg - Immunoglobulin\u003c/p\u003e\u003cp\u003eRBD - Receptor-binding domain\u003c/p\u003e\u003cp\u003eSARS-CoV-2 - Severe acute respiratory syndrome coronavirus 2\u003c/p\u003e\u003cp\u003eCOVID-19 - Coronavirus disease 2019\u003c/p\u003e\u003cp\u003eELISA - \u0026nbsp;Enzyme-linked immunosorbent assay\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch4\u003eEthics approval and consent to participate\u003c/h4\u003e\u003cp\u003eThis study was registered and approved by the institutional ethics committee of Adolfo Lutz Institute (CAAE 31924420.8.0000.0059), and written informed consent was provided by all participants. Additionally, all experiments were performed following relevant guidelines and regulations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThis study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP (grants numbers 2018/04202-0, 2018/14384-9, 2021/11936-3, 2022/05566-1); Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq (grants numbers 305301/2022-5, MS-DIAHV 24/2019 process 442776/2019-5, 132059/2025-8); Financiadora de Estudos e Projetos (Grant Number 01160075); Fundo de Investimento para Educação Sanitária e Imunização em Massa contra Doenças Transmissíveis/FESIMA (Grants Numbers 011/2021, 59/2021) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/CAPES (finance code 001).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eConceptualization and study design were performed by GSO, AIP, and EDG. Volunteer recruitment and data collection were performed by VOS and LFMB. Antigen production was carried out by HHMC and CRP. Laboratory experiments were performed by GSO, AIP, AFS, NSM, and EDG. Sample management was conducted by VOS and LFMB. Statistical analysis was performed by GSO and AIP. The manuscript was written by GSO, AIP, AFS, and NSM, and EDG revised the original draft. All authors contributed to the study, commented on previous versions of the manuscript, and approved the final manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors would like to thank Dr Florian Krammer (Mount Sinai Hospital, New York, NY, USA) for donating the plasmid to produce the RBD to Dr Carlos Roberto Prudencio.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ede la Poza, J.F.D., Par\u0026eacute;s, A.R., Aparicio-Calvente, I. et al. Frequency of IgE antibody response to SARS-CoV-2 RBD protein across different disease severity COVID19 groups. Virol J 22, 58 (2025). doi: 10.1186/s12985-025-02677-y.\u003c/li\u003e\n \u003cli\u003eHeyman B. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu Rev Immunol. 2000;18:709-737. doi:10.1146/annurev.immunol.18.1.709.\u003c/li\u003e\n \u003cli\u003eDa\u0026euml;ron M. The function of antibodies. Immunol Rev. 2024 Nov;328(1):113-125. doi: 10.1111/imr.13387.\u003c/li\u003e\n \u003cli\u003eKhan SR, Chaker L, Ikram MA, Peeters RP, van Hagen PM, Dalm VASH. Determinants and Reference Ranges of Serum Immunoglobulins in Middle-Aged and Elderly Individuals: a Population-Based Study. J Clin Immunol. 2021 Nov;41(8):1902-1914. doi: 10.1007/s10875-021-01120-5.\u003c/li\u003e\n \u003cli\u003eMoura, AD, da Costa, HHM, Correa, VA, Lima, AKS, Lindoso, JAL, Gaspari, ED, et al. Serological assessment of SARS-CoV-2 infection and vaccination using ELISA-based assays. Sci Rep 11 , 17642 (2021). doi: 10.1038/s41598-021-95045-z.\u003c/li\u003e\n \u003cli\u003ePlūme J, Galvanovskis A, \u0026Scaron;mite S, Romanchikova N, Zayakin P, Linē A. Early and strong antibody responses to SARS-CoV-2 predict disease severity in COVID-19 patients. J Transl Med. 2022 Apr 15;20(1):176. doi: 10.1186/s12967-022-03382-y.\u003c/li\u003e\n \u003cli\u003ePortilho AI, Silva VO, Da Costa HHM, Yamashiro R, de Oliveira IP, de Campos IB, et al. An unexpected IgE anti\u0026ndash;receptor-binding domain response following natural infection and different types of SARS-CoV-2 vaccine. Sci Rep. 2024 Aug 28;14(1):20003. doi: 10.1038/s41598-024-71047-5.\u003c/li\u003e\n \u003cli\u003eStadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, et al. SARS-CoV-2 seroconversion in humans: a detailed protocol for a serological assay. Curr Protoc Microbiol. 2020 Jun;57(1):e100. Doi: 10.1002/cpmc.100.\u003c/li\u003e\n \u003cli\u003eda Costa HHM, Silva VO, Amorim GC, Guereschi MG, Sergio LM, Gomes CHR, et al. Assessment of an in-house IgG ELISA targeting SARS-CoV-2 RBD for detection of antibody responses. J Immunol Methods. 2024 Jul;530:113683. doi: 10.1016/j.jim.2024.113683.\u003c/li\u003e\n \u003cli\u003eCrowther JR. The ELISA guidebook. 2nd ed. Methods in Molecular Biology. Totowa (NJ): Humana Press; 2009. doi:10.1007/978-1-60327-254-4.\u003c/li\u003e\n \u003cli\u003eKiszel P, S\u0026iacute;k P, Mikl\u0026oacute;s J, Kajd\u0026aacute;csi E, Sinkovits G, Cervenak L, et al. Class switch towards spike protein-specific IgG4 antibodies after SARS-CoV-2 mRNA vaccination depends on prior infection history. Sci Rep. 2023 Aug 13;13(1):13166. doi: 10.1038/s41598-023-40103-x.\u003c/li\u003e\n \u003cli\u003eIrrgang P, Gerling J, Kocher K, Lapuente D, Steininger P, Habenicht K, et al. Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination. Sci Immunol. 2023 Jan 27;8(79):eade2798. doi: 10.1126/sciimmunol.ade2798.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Instituto Adolfo Lutz","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"SARS-CoV-2, RBD, IgE, IgG4, indirect ELISA, assay sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-9031844/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9031844/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe detection of IgE antibodies specific to the receptor-binding domain (RBD) of SARS-CoV-2 has been associated with aspects of COVID-19 severity, including potential mast cell–mediated mechanisms. Recent studies analyzed IgE anti-RBD across different COVID-19 severity groups using ELISA. The coating-antigen concentration influences the detection capacity of the immunoassay; thus, low-abundance antibodies, such as IgE, may require higher antigen concentrations to achieve optimal experimental conditions. Here, we present the effect of different coating concentrations for anti-RBD IgE and IgG4 detection in samples of vaccinated individuals. Our results suggested that intermediate antigen concentrations provide a better balance between sensitivity and specificity, highlighting the importance of methodological standardization.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Challenges and Methodological Aspects of Detecting Anti-RBD Antibodies by ELISA in SARS-CoV-2 Research","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 10:37:40","doi":"10.21203/rs.3.rs-9031844/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":"ebbc4461-f9c0-4eac-8c44-38bb9778fe22","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63927899,"name":"Immunology"}],"tags":[],"updatedAt":"2026-03-05T10:37:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 10:37:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9031844","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9031844","identity":"rs-9031844","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}