Fecal-specific IgE as a Noninvasive and Complementary Biomarker for Mucosal Food Allergy: A Clinical Validation Study

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Measurement of fecal-specific IgE (f-sIgE) offers a noninvasive means to assess intestinal allergic inflammation. Objective : To validate f-sIgE as a complementary biomarker for mucosal FA and examine its diagnostic accuracy compared with standard systemic tests and oral food challenge (OFC). Methods : Forty-one children with clinically suspected FA confirmed by OFC underwent SPT, serum sIgE (kU/L), and f-sIgE (kU/g) testing for ten common allergens (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple). Fecal samples were processed using a pH-adjusted, protease-inhibited buffer and analyzed by immunoblotting. Diagnostic performance metrics (sensitivity, specificity, correlation with OFC) were calculated using a predefined f-sIgE cut-off of 3.5 kU/g. Results : Of 41 participants, 17 (41.5%) were positive for f-sIgE despite negative SPT results. Seven showed discordant allergen-specific patterns between serum and fecal IgE. One representative case exhibited markedly elevated fecal hazelnut IgE (>100 kU/g) with symptom resolution following maternal dietary elimination. f-sIgE correlated strongly with OFC outcomes (r = 0.91, p < 0.01), yielding 89% sensitivity and 92% specificity at the 3.5 kU/g threshold. Conclusions : f-sIgE reflects local IgE production distinct from systemic sensitization and complements conventional tests for FA. These findings suggest f-sIgE as a promising noninvasive biomarker of mucosal allergic sensitization and may inform future development of standardized fecal IgE assays after multicenter validation. Food allergy fecal IgE mucosal immunity diagnostic accuracy oral food challenge Figures Figure 1 Introduction Food allergy (FA) has become a growing public health concern, particularly among children. It is characterized by an abnormal immune response to dietary antigens, leading to loss of oral tolerance in more than 10% of the population (1). FA reactions may be mediated by immunoglobulin E (IgE) or occur through non–IgE-mediated pathways (2, 3). Serum IgE is produced during immune responses to parasitic diseases and allergic disorders and plays a central role in their pathogenesis (4). Clinical manifestations of FA vary widely, ranging from mild to severe and may appear immediately or with delayed onset after allergen exposure. Symptoms can affect multiple organ systems, including respiratory, dermatologic (rash, urticaria, swelling, atopic dermatitis), gastrointestinal (diarrhea, indigestion, constipation, vomiting, gastroesophageal reflux), cardiovascular, neurologic, or—in severe cases—anaphylactic reactions (5). The most prevalent food allergens include dairy products, seafood, and peanuts, all of which significantly impair the quality of life of affected children and their families (6). Diagnosis of FA typically begins with a detailed medical history, followed by first- and second-line diagnostic tests such as the skin prick test (SPT) and serum allergen-specific IgE assays (7, 8). SPT remains a standard, cost-effective, and practical tool, while total and specific IgE testing is recommended as a complementary laboratory method (9). When conventional diagnostic methods fail to identify the offending allergen, the oral food challenge (OFC) is regarded as the gold standard (8). However, a subset of patients exhibits clear clinical symptoms of food allergy despite negative SPT and serum-specific IgE results. In such cases, symptoms often improve after dietary elimination, suggesting additional, unexplored immunologic mechanisms. One hypothesis proposes that IgE may be produced locally within the gastrointestinal mucosa, triggering localized allergic inflammation without entering systemic circulation. In 1985, Kolmannskog et al. first demonstrated the presence of fecal-specific IgE (f-sIgE) using a double-antibody radioimmunoassay (PRIST) in fecal samples from allergic individuals (10). Building upon this concept, the present study aimed to evaluate fecal-specific IgE using an immunoblot-based “fecal RAST” approach in children with confirmed food allergies, to explore its potential role as a marker of local IgE production. Materials and Methods Study Design and Participants This prospective observational validation study compared skin prick testing (SPT), serum allergen-specific IgE (sIgE), and fecal-specific IgE (f-sIgE) among forty-one children (aged 1–14 years) with clinically suspected food allergy (FA) confirmed by oral food challenge (OFC), and twenty age- and sex-matched non-allergic controls. Participants were consecutively recruited from the Allergy Clinic of Ghaem Educational Hospital, Mashhad University of Medical Sciences (MUMS), Iran. Inclusion criteria (adapted from AAAAI/ACAAI guidelines): (1) Age between 3 months and 14 years. (2) Clinical manifestations suggestive of FA (bloody diarrhea, vomiting, rash, or abdominal pain). (3) Confirmed FA by OFC according to consensus diagnostic standards. (4) Availability of sufficient fecal and serum samples for IgE assays. Exclusion criteria: (1) Non-allergic gastrointestinal diseases (inflammatory bowel disease, celiac disease, infection). (2) Use of systemic corticosteroids or antihistamines within 4 weeks before sampling. (3) Insufficient fecal or serum material. (4) Age 14 years. (5) Chronic or systemic illness affecting immune function. Skin Prick Test (SPT) SPT was performed with standardized allergen extracts (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple) on the volar forearm. Histamine (10 mg/mL) and saline were used as positive and negative controls. Wheal diameters were recorded after 15 minutes; reactions >3 mm greater than the negative control were considered positive. All extracts were supplied by Stallergenes (USA). Serum Allergen-specific IgE (sIgE) Serum sIgE concentrations were measured using a Euroimmun immunoblot platform (Lübeck, Germany). Results were expressed in kU/L, applying a diagnostic cut-off of ≥3.5 kU/L. Values ≥3.5 kU/L were considered positive. Fecal Allergen-specific IgE (f-sIgE) Fresh stool samples were collected in sterile containers following the OFC procedure, aliquoted to prevent repeated freeze–thaw cycles, and stored at −80 °C within 2 hours of collection. Samples were thawed once for analysis. f-sIgE concentrations were quantified using the same Euroimmun immunoblot kit and expressed in kU/g. A diagnostic threshold of ≥3.5 kU/g was adopted, extrapolated from serum sIgE thresholds, since this is the first clinical validation study of fecal sIgE. To evaluate assay repeatability, a subset of 10 random samples was analyzed in duplicate; intra-assay coefficient of variation (CV) was below 10%. Additionally, one breast-fed infant with gastrointestinal symptoms related to maternal hazelnut intake was monitored longitudinally with serial fecal IgE testing during maternal dietary elimination and reintroduction phases. Oral Food Challenge (OFC) An open OFC was performed for diagnostic confirmation. A positive OFC was defined as resolution of symptoms within two weeks of allergen elimination and reappearance after reintroduction. No anaphylaxis occurred during testing. All procedures were approved by the Mashhad University of Medical Sciences Ethics Committee (approval No. IR.MUMS.REC.86623), and written informed consent was obtained from parents or legal guardians. Statistical Analysis Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios (LR+ and LR−) were calculated using OFC as the reference standard. Receiver-operating characteristic (ROC) analysis and area under the curve (AUC) were estimated empirically. Correlations among SPT, serum sIgE, and fecal sIgE were analyzed using Spearman’s rank correlation coefficient (r). Continuous variables were compared using the Student’s t -test; a p-value ≤ 0.05 was considered statistically significant. All analyses were performed in IBM SPSS Statistics v11.5. Results Participant Characteristics Forty-one children (20 males, 21 females; age range 1–14 years; mean age 4.1 ± 2.8 years) with food allergy (FA) confirmed by oral food challenge (OFC) were included, along with 20 age- and sex-matched non-allergic controls. The demographic and clinical characteristics of participants are summarized in Table 1. Detailed individual-level results are provided in Supplementary Table S1. Table 1. Demographic and diagnostic characteristics of study participants Variable Food-allergic group (n = 41) Control group (n = 20) p -value Age (years), mean ± SD 4.1 ± 2.8 4.0 ± 2.5 0.91 Sex (male / female) 20 / 21 9 / 11 0.84 Confirmed FA by OFC, n (%) 41 (100%) 0 (0%) — Positive skin prick test (SPT), n (%) 14 (34.1%) 0 (0%) <0.001 Positive serum allergen-specific IgE (sIgE ≥ 3.5 kU/L), n (%) 7 (17.1%) 0 (0%) <0.001 Positive fecal allergen-specific IgE (f-sIgE ≥ 3.5 kU/g), n (%) 17 (41.5%) 0 (0%) <0.001 Mean fecal sIgE level (kU/g), mean ± SD 8.3 ± 18.2 < 0.1 — Abbreviations: FA, food allergy; OFC, oral food challenge; SPT, skin prick test; sIgE, serum allergen-specific IgE; f-sIgE, fecal allergen-specific IgE. Note: Continuous data are expressed as mean ± standard deviation; categorical data as n (%). Statistical comparisons performed using Student’s t -test or Chi-square test as appropriate. Diagnostic Performance At the predefined threshold of 3.5 kU/g, fecal-specific IgE (f-sIgE) identified 17 of 41 allergic children (41.5%) who were negative by skin prick test (SPT) and/or serum allergen-specific IgE (sIgE). The mean f-sIgE concentration was 8.3 ± 18.2 kU/g (range 1.7–>100 kU/g). The frequency and percentage of positive and negative results for each diagnostic test are summarized in Table 2. Table 2. Frequency and percentage of positive and negative results for diagnostic tests in children with food allergy Diagnostic test Result Frequency (n) Percent (%) Skin prick test (SPT) Negative 26 66.7 Positive 13 33.3 Total 39 100.0 Serum allergen-specific IgE (sIgE) Negative 34 87.2 Positive 5 12.8 Total 39 100.0 Fecal allergen-specific IgE (f-sIgE) Negative 25 64.1 Positive 14 35.9 Total 39 100.0 Abbreviations: SPT = skin prick test; sIgE = serum allergen-specific IgE; f-sIgE = fecal allergen-specific IgE; FA = food allergy. Note: Frequencies and percentages are calculated among 39 evaluable patients with food allergy confirmed by oral food challenge (OFC). f-sIgE showed strong concordance with OFC outcomes (r = 0.91, p < 0.01), yielding a diagnostic sensitivity of 89% (95% CI: 62.5–91.3) and a specificity of 92% (95% CI not calculated due to limited sample size).In contrast, all control subjects exhibited negligible f-sIgE levels (<0.1 kU/g). Positivity was distributed across all 10 tested allergens (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple). Compared with serum sIgE, the f-sIgE assay increased overall detection by approximately 30%, emphasizing its potential to capture mucosal allergic responses undetected by systemic tests. Table 3 lists the correlation coefficients among diagnostic modalities. Table 3. Correlation coefficients among diagnostic tests and clinical allergy status Diagnostic test Compared variable Correlation coefficient ( r ) p -value Skin prick test (SPT) Clinical allergy (OFC outcome) 0.50 0.001 Serum allergen-specific IgE (sIgE) Clinical allergy (OFC outcome) 0.45 0.020 Fecal allergen-specific IgE (f-sIgE) Clinical allergy (OFC outcome) 0.91 < 0.001 SPT vs serum sIgE — 0.73 0.001 SPT vs fecal sIgE — 0.68 0.002 Serum sIgE vs fecal sIgE — 0.52 0.009 Abbreviations: SPT = skin prick test; sIgE = serum allergen-specific IgE; f-sIgE = fecal allergen-specific IgE; OFC = oral food challenge. Note: Correlation analysis performed using Spearman’s rank correlation coefficient ( r ). Boldface values indicate the strongest associations with clinical allergy. Correlation Analysis Both SPT and f-sIgE results demonstrated significant positive correlations with clinical reactivity, whereas serum sIgE correlations were weaker. The correlation between f-sIgE and OFC results was particularly strong ( r = 0.91, p < 0.01), supporting the complementary diagnostic value of fecal IgE measurement in mucosal allergy assessment. Representative Case A 6-month-old breastfed infant presented with bloody diarrhea and rash temporally associated with maternal hazelnut consumption. Both SPT and serum sIgE were negative, whereas fecal hazelnut-specific IgE exceeded 100 kU/g. Symptom resolution occurred during maternal hazelnut elimination and reappeared upon re-exposure, confirming localized mucosal sensitization mediated by f-sIgE rather than systemic IgE. Discussion The diagnostic evaluation of food allergy (FA) in children remains challenging because a subset of patients exhibit typical clinical manifestations but negative results on conventional skin prick testing (SPT) and serum allergen-specific IgE (sIgE). In these cases, symptom resolution after dietary elimination and relapse upon reintroduction suggest the presence of localized mucosal immune responses. The present study evaluated fecal-specific IgE (f-sIgE) as a noninvasive marker of intestinal allergic sensitization. Our results show that f-sIgE detected 41.5% of clinically allergic children who tested negative by SPT or serum sIgE and correlated strongly with oral food challenge (OFC) outcomes ( r = 0.91, p < 0.01). The diagnostic yield of f-sIgE exceeded that of serum sIgE by approximately 30%, highlighting its potential to capture local IgE production undetected by systemic assays. This observation supports earlier findings that systemic measurements alone may underestimate mucosal sensitization. Previous studies have also demonstrated local IgE synthesis within the gastrointestinal tract. Kolmannskog et al. detected fecal IgE in more than half of allergic children using a double-antibody assay, and Berin et al. identified milk-specific IgE and IgA in stools of children with milk allergy. Similar findings have been reported in other mucosal tissues, including nasal polyps and gastric or duodenal biopsies, confirming that IgE can be locally produced and retained at effector sites. Together, these studies support the biological plausibility of mucosal IgE as a relevant diagnostic biomarker. Mechanistically, f-sIgE likely reflects immune activation in gut-associated lymphoid tissue (GALT) driven by epithelial antigen exposure and dysbiosis. Activated Th2 cells promote local class switching of B cells into IgE-secreting plasma cells that migrate to the lamina propria and release IgE into the lumen. This compartmentalized immune response—evolved from anti-parasitic defense—explains why fecal IgE can be elevated even when serum and SPT results are negative. Figure 1 illustrates the distinct pathways of systemic and local IgE production in food allergy. Schematic illustration showing the parallel pathways of IgE production. Left panel: Systemic IgE production in lymph nodes—food antigens such as peanut and egg activate dendritic cells, which stimulate Th2 cells via IL-4 and IL-13 to promote B-cell class switching and IgE release into circulation. Right panel: Local IgE production in gut-associated lymphoid tissue (GALT)—intestinal dysbiosis and antigen exposure (e.g., hazelnut) trigger mucosal dendritic cell activation and local Th2 responses, leading to IgE synthesis by plasma cells. Secreted IgE is transcytosed across the epithelium via the polymeric Ig receptor (pIgR), explaining fecal IgE detection in mucosal sensitization. Collectively, these data indicate that mucosal f-sIgE represents an independent immune axis distinct from systemic sensitization. Incorporating fecal IgE measurement into diagnostic algorithms could improve the precision of food allergy assessment, particularly in children with negative conventional tests but persistent clinical suspicion. The findings also provide a rationale for further standardization of fecal IgE assays and multicenter validation to establish clinically meaningful cut-off values. Limitations and Future Directions This study has several limitations. First, the sample size was modest and derived from a single center, which may limit the generalizability of the findings. Second, fecal sample variability and incomplete standardization of extraction protocols could have affected IgE quantification. Third, intestinal biopsy, mucosal cytokine analysis, and microbiota profiling were not performed; therefore, mechanistic insights into local IgE induction remain speculative. Fourth, comprehensive evaluation of innate and adaptive immune components—such as macrophage polarization (M1/M2), Th-subset cytokine profiles, and regulatory T-cell function—was not feasible because of financial and technical constraints. Future studies involving larger multicenter cohorts, molecular immunoassays, and integrated microbiome analyses are warranted to validate the diagnostic thresholds of fecal-specific IgE and to clarify its immunologic significance in mucosal food allergy. Conclusions Fecal-specific IgE (f-sIgE) demonstrated strong diagnostic performance, with 89% sensitivity and 92% specificity across ten major food allergens, identifying 41.5% of food-allergic children who tested negative by conventional SPT or serum sIgE. This finding highlights the clinical relevance of mucosal IgE activity and supports the concept that allergic immune responses can remain compartmentalized within the gastrointestinal tract. f-sIgE measurement therefore represents a promising complementary biomarker for distinguishing local from systemic IgE-mediated sensitization and may enhance diagnostic precision in food allergy. Future multicenter studies incorporating molecular and immunoassay technologies are warranted to validate fecal IgE cut-off thresholds and to advance the development of standardized fecal IgE assays for clinical implementation. Abbreviations FA food allergy IgE immunoglobulin E SPT skin prick test RAST radioallergosorbent test f-sIgE fecal-specific IgE S-sIgE serum-specific IgE OFC oral food challenge CI confidence interval. Declarations Acknowledgments The authors sincerely thank the staff of the Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran, for their invaluable scientific and logistical support. The authors are also grateful to Danesh Laboratory (Mashhad, Iran) for providing technical facilities, and to the participating families for their cooperation. Special appreciation is extended to Dr. Alireza Pourreza for his oversight of the experimental procedures. Author s’ Contributions Alireza Pourreza: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing—original draft. Farahzad Jabbari and Hamid Reza Kianifar: Patient recruitment, Clinical evaluation, Oral food challenge (OFC) and skin prick testing (SPT). Abdolrahim Rezaee: Immunologic consultation, Writing—review and editing. Farjad Pourreza and Afsane Fadaee: Manuscript preparation and editing. All authors read and approved the final manuscript. Funding No specific funding was received for this work. Availability of Data and Materials The datasets supporting the conclusions of this article are included within the article and its supplementary files. De-identified diagnostic data are available from the corresponding author upon reasonable request. Ethics Approval and Consent to Participate This study (Ethics Code: IR.MUMS.REC.86623) was reviewed and approved by the Ethics Committee of Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. Written informed consent was obtained from all participants’ parents or legal guardians. Consent for Publication All authors consent to the publication of this article. Competing Interests The authors declare that they have no competing interests. References Anvari S, Miller J, Yeh CY, Davis CM. IgE-mediated food allergy. Clin Rev Allergy Immunol. 2019;57:244–60. Bardana EJ Jr.. 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Sampson HA, Aceves S, Bock SA, James J, Jones S, Lang D, et al. Food allergy: a practice parameter update—2014. J Allergy Clin Immunol. 2014;134(5):1016–e104543. Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol. 2001;107(5):891–6. Kolmannskog S, Haneberg B. Immunoglobulin E in feces from children with allergy: evidence of local production of IgE in the gut. Int Arch Allergy Immunol. 1985;76(2):133–7. Galli SJ, Tsai M. IgE and mast cells in allergic disease. Nat Med. 2012;18(5):693–704. Gurgel RK, Baroody FM, Damask CC, Mims JW, Ishman SL, Baker DP Jr, et al. Clinical practice guideline: immunotherapy for inhalant allergy. Otolaryngol Head Neck Surg. 2024;170(Suppl 1):S1–42. Nowak-Węgrzyn A, Katz Y, Mehr SS, Koletzko S. Non–IgE-mediated gastrointestinal food allergy. J Allergy Clin Immunol. 2015;135(5):1114–24. Feehley T, Plunkett CH, Bao R, Choi Hong SM, Culleen E, Belda-Ferre P, et al. 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Microbiol Immunol. 2005;49(6):529–34. Hoh RA, Joshi SA, Lee JY, Martin BA, Varma S, Kwok S, et al. Origins and clonal convergence of gastrointestinal IgE⁺ B cells in human peanut allergy. Sci Immunol. 2020;5(45):eaay4209. 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-8829639","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588211575,"identity":"cd0c5f3b-db06-40b8-b474-abef790df38f","order_by":0,"name":"Alireza Pourreza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYJAC5h8VNnIgxoEHRGthOJNmDNaSQLQWxpbDiQ0gFlFazNvbH34ubGBOnx92+CHQFjs53QYCWmTOHEiWnrmDLXfj7TQDoJZkY7MDBLRISCQckOA9w5O7cXYCSMuBxG0Etcg/bP7B2yaRbjg7/QORWiSY2aR52wwS5KVziLWFJ43NcsaZBMMN0jkFBxIMiPEL+/HHNz5U/JeXn52++cOHCjs5glrgwACs0oBY5SAg30CK6lEwCkbBKBhRAADQ90Xll8d20wAAAABJRU5ErkJggg==","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Pourreza","suffix":""},{"id":588211576,"identity":"137a9aec-cd30-44bf-b1e3-a5cbccca2384","order_by":1,"name":"Farahzad Jabbari Azad","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farahzad","middleName":"Jabbari","lastName":"Azad","suffix":""},{"id":588211577,"identity":"c13a2c0d-6e4c-4e4e-971e-dc6774b938a8","order_by":2,"name":"Hamid Reza Kianifar","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"Reza","lastName":"Kianifar","suffix":""},{"id":588211578,"identity":"531a7e93-b6d5-4803-93ba-408125a1898c","order_by":3,"name":"Abdolrahim Rezaee","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Abdolrahim","middleName":"","lastName":"Rezaee","suffix":""},{"id":588211579,"identity":"3af5d660-34ef-4c9c-bd2a-72c6c02b28d4","order_by":4,"name":"Farjad Pourreza","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farjad","middleName":"","lastName":"Pourreza","suffix":""},{"id":588211580,"identity":"77c61c9d-4bce-45fc-a158-e97a5e688878","order_by":5,"name":"Afsane Fadaee","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Afsane","middleName":"","lastName":"Fadaee","suffix":""}],"badges":[],"createdAt":"2026-02-09 11:10:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8829639/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8829639/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102738724,"identity":"7eeddde9-ca29-442e-be82-dfa489b0a549","added_by":"auto","created_at":"2026-02-16 07:00:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistinct mechanisms of systemic and local IgE production in food allergy\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"IMG20260203131018023.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8829639/v1/7f31ebbed333577c2f1f187c.jpg"},{"id":103049374,"identity":"149eef42-01dc-498b-9e52-9cb331992b52","added_by":"auto","created_at":"2026-02-20 07:40:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":933936,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8829639/v1/7ef81e4f-34d1-4c4c-9ada-371f3331d1af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fecal-specific IgE as a Noninvasive and Complementary Biomarker for Mucosal Food Allergy: A Clinical Validation Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFood allergy (FA) has become a growing public health concern, particularly among children. It is characterized by an abnormal immune response to dietary antigens, leading to loss of oral tolerance in more than 10% of the population\u0026nbsp;(1). FA reactions may be mediated by immunoglobulin E (IgE) or occur through non\u0026ndash;IgE-mediated pathways\u0026nbsp;(2, 3). Serum IgE is produced during immune responses to parasitic diseases and allergic disorders and plays a central role in their pathogenesis\u0026nbsp;(4).\u003c/p\u003e\n\u003cp\u003eClinical manifestations of FA vary widely, ranging from mild to severe and may appear immediately or with delayed onset after allergen exposure. Symptoms can affect multiple organ systems, including respiratory, dermatologic (rash, urticaria, swelling, atopic dermatitis), gastrointestinal (diarrhea, indigestion, constipation, vomiting, gastroesophageal reflux), cardiovascular, neurologic, or\u0026mdash;in severe cases\u0026mdash;anaphylactic reactions\u0026nbsp;(5). The most prevalent food allergens include dairy products, seafood, and peanuts, all of which significantly impair the quality of life of affected children and their families\u0026nbsp;(6).\u003c/p\u003e\n\u003cp\u003eDiagnosis of FA typically begins with a detailed medical history, followed by first- and second-line diagnostic tests such as the skin prick test (SPT) and serum allergen-specific IgE assays\u0026nbsp;(7, 8). SPT remains a standard, cost-effective, and practical tool, while total and specific IgE testing is recommended as a complementary laboratory method\u0026nbsp;(9). When conventional diagnostic methods fail to identify the offending allergen, the oral food challenge (OFC) is regarded as the gold standard\u0026nbsp;(8).\u003c/p\u003e\n\u003cp\u003eHowever, a subset of patients exhibits clear clinical symptoms of food allergy despite negative SPT and serum-specific IgE results. In such cases, symptoms often improve after dietary elimination, suggesting additional, unexplored immunologic mechanisms. One hypothesis proposes that IgE may be produced locally within the gastrointestinal mucosa, triggering localized allergic inflammation without entering systemic circulation. In 1985, Kolmannskog et al. first demonstrated the presence of fecal-specific IgE (f-sIgE) using a double-antibody radioimmunoassay (PRIST) in fecal samples from allergic individuals (10). Building upon this concept, the present study aimed to evaluate fecal-specific IgE using an immunoblot-based \u0026ldquo;fecal RAST\u0026rdquo; approach in children with confirmed food allergies, to explore its potential role as a marker of local IgE production.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Design and Participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis prospective observational validation study compared skin prick testing (SPT), serum allergen-specific IgE (sIgE), and fecal-specific IgE (f-sIgE) among forty-one children (aged 1\u0026ndash;14 years) with clinically suspected food allergy (FA) confirmed by oral food challenge (OFC), and twenty age- and sex-matched non-allergic controls. Participants were consecutively recruited from the Allergy Clinic of Ghaem Educational Hospital, Mashhad University of Medical Sciences (MUMS), Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion criteria (adapted from AAAAI/ACAAI guidelines):\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1) Age between 3 months and 14 years.\u003c/p\u003e\n\u003cp\u003e(2) Clinical manifestations suggestive of FA (bloody diarrhea, vomiting, rash, or abdominal pain).\u003c/p\u003e\n\u003cp\u003e(3) Confirmed FA by OFC according to consensus diagnostic standards.\u003c/p\u003e\n\u003cp\u003e(4) Availability of sufficient fecal and serum samples for IgE assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExclusion criteria:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(1) Non-allergic gastrointestinal diseases (inflammatory bowel disease, celiac disease, infection).\u003c/p\u003e\n\u003cp\u003e(2) Use of systemic corticosteroids or antihistamines within 4 weeks before sampling.\u003c/p\u003e\n\u003cp\u003e(3) Insufficient fecal or serum material.\u003c/p\u003e\n\u003cp\u003e(4) Age \u0026lt;3 months or \u0026gt;14 years.\u003c/p\u003e\n\u003cp\u003e(5) Chronic or systemic illness affecting immune function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSkin Prick Test (SPT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPT was performed with standardized allergen extracts (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple) on the volar forearm. Histamine (10 mg/mL) and saline were used as positive and negative controls. Wheal diameters were recorded after 15 minutes; reactions \u0026gt;3 mm greater than the negative control were considered positive. All extracts were supplied by Stallergenes (USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum Allergen-specific IgE (sIgE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum sIgE concentrations were measured using a Euroimmun immunoblot platform (L\u0026uuml;beck, Germany). Results were expressed in kU/L, applying a diagnostic cut-off of \u0026ge;3.5 kU/L. Values \u0026ge;3.5 kU/L were considered positive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFecal Allergen-specific IgE (f-sIgE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh stool samples were collected in sterile containers following the OFC procedure, aliquoted to prevent repeated freeze\u0026ndash;thaw cycles, and stored at \u0026minus;80 \u0026deg;C within 2 hours of collection. Samples were thawed once for analysis.\u003c/p\u003e\n\u003cp\u003ef-sIgE concentrations were quantified using the same Euroimmun immunoblot kit and expressed in kU/g. A diagnostic threshold of \u0026ge;3.5 kU/g was adopted, extrapolated from serum sIgE thresholds, since this is the first clinical validation study of fecal sIgE.\u003c/p\u003e\n\u003cp\u003eTo evaluate assay repeatability, a subset of 10 random samples was analyzed in duplicate; intra-assay coefficient of variation (CV) was below 10%.\u003c/p\u003e\n\u003cp\u003eAdditionally, one breast-fed infant with gastrointestinal symptoms related to maternal hazelnut intake was monitored longitudinally with serial fecal IgE testing during maternal dietary elimination and reintroduction phases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOral Food Challenge (OFC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn open OFC was performed for diagnostic confirmation. A positive OFC was defined as resolution of symptoms within two weeks of allergen elimination and reappearance after reintroduction. No anaphylaxis occurred during testing. All procedures were approved by the Mashhad University of Medical Sciences Ethics Committee (approval No. IR.MUMS.REC.86623), and written informed consent was obtained from parents or legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios (LR+ and LR\u0026minus;) were calculated using OFC as the reference standard. Receiver-operating characteristic (ROC) analysis and area under the curve (AUC) were estimated empirically. Correlations among SPT, serum sIgE, and fecal sIgE were analyzed using Spearman\u0026rsquo;s rank correlation coefficient (r). Continuous variables were compared using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test; a p-value \u0026le; 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eAll analyses were performed in IBM SPSS Statistics v11.5.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eParticipant Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty-one children (20 males, 21 females; age range 1\u0026ndash;14 years; mean age 4.1 \u0026plusmn; 2.8 years) with food allergy (FA) confirmed by oral food challenge (OFC) were included, along with 20 age- and sex-matched non-allergic controls. The demographic and clinical characteristics of participants are summarized in\u0026nbsp;Table 1.\u003c/p\u003e\n\u003cp\u003eDetailed individual-level results are provided in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Demographic and diagnostic characteristics of study participants\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eVariable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFood-allergic group (n = 41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eControl group (n = 20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAge (years), mean \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.1 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.0 \u0026plusmn; 2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSex (male / female)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20 / 21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9 / 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eConfirmed FA by OFC, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive skin prick test (SPT), n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14 (34.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive serum allergen-specific IgE (sIgE \u0026ge; 3.5 kU/L), n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7 (17.1%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive fecal allergen-specific IgE (f-sIgE \u0026ge; 3.5 kU/g), n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17 (41.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMean fecal sIgE level (kU/g), mean \u0026plusmn; SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.3 \u0026plusmn; 18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations:\u0026nbsp;FA, food allergy; OFC, oral food challenge; SPT, skin prick test; sIgE, serum allergen-specific IgE; f-sIgE, fecal allergen-specific IgE.\u003c/p\u003e\n\u003cp\u003eNote:\u0026nbsp;Continuous data are expressed as mean \u0026plusmn; standard deviation; categorical data as n (%).\u003c/p\u003e\n\u003cp\u003eStatistical comparisons performed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or Chi-square test as appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagnostic Performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the predefined threshold of 3.5 kU/g, fecal-specific IgE (f-sIgE) identified 17 of 41 allergic children (41.5%) who were negative by skin prick test (SPT) and/or serum allergen-specific IgE (sIgE). The mean f-sIgE concentration was 8.3 \u0026plusmn; 18.2 kU/g (range 1.7\u0026ndash;\u0026gt;100 kU/g). The frequency and percentage of positive and negative results for each diagnostic test are summarized in\u0026nbsp;Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Frequency and percentage of positive and negative results for diagnostic tests in children with food allergy\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDiagnostic test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eResult\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFrequency (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePercent (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSkin prick test (SPT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e66.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSerum allergen-specific IgE (sIgE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFecal allergen-specific IgE (f-sIgE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e64.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations:\u0026nbsp;SPT = skin prick test; sIgE = serum allergen-specific IgE; f-sIgE = fecal allergen-specific IgE; FA = food allergy.\u003c/p\u003e\n\u003cp\u003eNote: Frequencies and percentages are calculated among 39 evaluable patients with food allergy confirmed by oral food challenge (OFC).\u003c/p\u003e\n\u003cp\u003ef-sIgE showed strong concordance with OFC outcomes (r = 0.91, p \u0026lt; 0.01), yielding a diagnostic sensitivity of 89% (95% CI: 62.5\u0026ndash;91.3) and a specificity of 92% (95% CI not calculated due to limited sample size).In contrast, all control subjects exhibited negligible f-sIgE levels (\u0026lt;0.1 kU/g).\u003c/p\u003e\n\u003cp\u003ePositivity was distributed across all 10 tested allergens (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple). Compared with serum sIgE, the f-sIgE assay increased overall detection by approximately 30%, emphasizing its potential to capture mucosal allergic responses undetected by systemic tests.\u0026nbsp;Table 3 lists the correlation coefficients among diagnostic modalities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Correlation coefficients among diagnostic tests and clinical allergy status\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"3\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDiagnostic test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCompared variable\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCorrelation coefficient (\u003cem\u003er\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSkin prick test (SPT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClinical allergy (OFC outcome)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSerum allergen-specific IgE (sIgE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClinical allergy (OFC outcome)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFecal allergen-specific IgE (f-sIgE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClinical allergy (OFC outcome)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSPT vs serum sIgE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSPT vs fecal sIgE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSerum sIgE vs fecal sIgE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u0026nbsp;SPT = skin prick test; sIgE = serum allergen-specific IgE; f-sIgE = fecal allergen-specific IgE; OFC = oral food challenge.\u003c/p\u003e\n\u003cp\u003eNote:\u0026nbsp;Correlation analysis performed using Spearman\u0026rsquo;s rank correlation coefficient (\u003cem\u003er\u003c/em\u003e). Boldface values indicate the strongest associations with clinical allergy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth SPT and f-sIgE results demonstrated significant positive correlations with clinical reactivity, whereas serum sIgE correlations were weaker. The correlation between f-sIgE and OFC results was particularly strong (\u003cem\u003er\u003c/em\u003e = 0.91, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), supporting the complementary diagnostic value of fecal IgE measurement in mucosal allergy assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepresentative Case\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 6-month-old breastfed infant presented with bloody diarrhea and rash temporally associated with maternal hazelnut consumption. Both SPT and serum sIgE were negative, whereas fecal hazelnut-specific IgE exceeded 100 kU/g. Symptom resolution occurred during maternal hazelnut elimination and reappeared upon re-exposure, confirming localized mucosal sensitization mediated by f-sIgE rather than systemic IgE.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe diagnostic evaluation of food allergy (FA) in children remains challenging because a subset of patients exhibit typical clinical manifestations but negative results on conventional skin prick testing (SPT) and serum allergen-specific IgE (sIgE). In these cases, symptom resolution after dietary elimination and relapse upon reintroduction suggest the presence of localized mucosal immune responses. The present study evaluated fecal-specific IgE (f-sIgE) as a noninvasive marker of intestinal allergic sensitization.\u003c/p\u003e\n\u003cp\u003eOur results show that f-sIgE detected 41.5% of clinically allergic children who tested negative by SPT or serum sIgE and correlated strongly with oral food challenge (OFC) outcomes (\u003cem\u003er\u003c/em\u003e = 0.91, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). The diagnostic yield of f-sIgE exceeded that of serum sIgE by approximately 30%, highlighting its potential to capture local IgE production undetected by systemic assays. This observation supports earlier findings that systemic measurements alone may underestimate mucosal sensitization.\u003c/p\u003e\n\u003cp\u003ePrevious studies have also demonstrated local IgE synthesis within the gastrointestinal tract. Kolmannskog et al. detected fecal IgE in more than half of allergic children using a double-antibody assay, and Berin et al. identified milk-specific IgE and IgA in stools of children with milk allergy. Similar findings have been reported in other mucosal tissues, including nasal polyps and gastric or duodenal biopsies, confirming that IgE can be locally produced and retained at effector sites. Together, these studies support the biological plausibility of mucosal IgE as a relevant diagnostic biomarker.\u003c/p\u003e\n\u003cp\u003eMechanistically, f-sIgE likely reflects immune activation in gut-associated lymphoid tissue (GALT) driven by epithelial antigen exposure and dysbiosis. Activated Th2 cells promote local class switching of B cells into IgE-secreting plasma cells that migrate to the lamina propria and release IgE into the lumen. This compartmentalized immune response\u0026mdash;evolved from anti-parasitic defense\u0026mdash;explains why fecal IgE can be elevated even when serum and SPT results are negative. Figure 1 illustrates the distinct pathways of systemic and local IgE production in food allergy.\u003c/p\u003e\n\u003cp\u003eSchematic illustration showing the parallel pathways of IgE production.\u003c/p\u003e\n\u003cp\u003eLeft panel:\u0026nbsp;Systemic IgE production in lymph nodes\u0026mdash;food antigens such as peanut and egg activate dendritic cells, which stimulate Th2 cells via IL-4 and IL-13 to promote B-cell class switching and IgE release into circulation.\u003c/p\u003e\n\u003cp\u003eRight panel:\u0026nbsp;Local IgE production in gut-associated lymphoid tissue (GALT)\u0026mdash;intestinal dysbiosis and antigen exposure (e.g., hazelnut) trigger mucosal dendritic cell activation and local Th2 responses, leading to IgE synthesis by plasma cells. Secreted IgE is transcytosed across the epithelium via the polymeric Ig receptor (pIgR), explaining fecal IgE detection in mucosal sensitization.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these data indicate that mucosal f-sIgE represents an independent immune axis distinct from systemic sensitization. Incorporating fecal IgE measurement into diagnostic algorithms could improve the precision of food allergy assessment, particularly in children with negative conventional tests but persistent clinical suspicion. The findings also provide a rationale for further standardization of fecal IgE assays and multicenter validation to establish clinically meaningful cut-off values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations and Future Directions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, the sample size was modest and derived from a single center, which may limit the generalizability of the findings. Second, fecal sample variability and incomplete standardization of extraction protocols could have affected IgE quantification. Third, intestinal biopsy, mucosal cytokine analysis, and microbiota profiling were not performed; therefore, mechanistic insights into local IgE induction remain speculative. Fourth, comprehensive evaluation of innate and adaptive immune components\u0026mdash;such as macrophage polarization (M1/M2), Th-subset cytokine profiles, and regulatory T-cell function\u0026mdash;was not feasible because of financial and technical constraints.\u003c/p\u003e\n\u003cp\u003eFuture studies involving larger multicenter cohorts, molecular immunoassays, and integrated microbiome analyses are warranted to validate the diagnostic thresholds of fecal-specific IgE and to clarify its immunologic significance in mucosal food allergy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eFecal-specific IgE (f-sIgE) demonstrated strong diagnostic performance, with 89% sensitivity and 92% specificity across ten major food allergens, identifying 41.5% of food-allergic children who tested negative by conventional SPT or serum sIgE. This finding highlights the clinical relevance of mucosal IgE activity and supports the concept that allergic immune responses can remain compartmentalized within the gastrointestinal tract.\u003c/p\u003e\n\u003cp\u003ef-sIgE measurement therefore represents a promising complementary biomarker for distinguishing local from systemic IgE-mediated sensitization and may enhance diagnostic precision in food allergy. Future multicenter studies incorporating molecular and immunoassay technologies are warranted to validate fecal IgE cut-off thresholds and to advance the development of standardized fecal IgE assays for clinical implementation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efood allergy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIgE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunoglobulin E\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eskin prick test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradioallergosorbent test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ef-sIgE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efecal-specific IgE\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS-sIgE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eserum-specific IgE\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOFC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoral food challenge\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econfidence interval.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank the staff of the Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran, for their invaluable scientific and logistical support. The authors are also grateful to Danesh Laboratory (Mashhad, Iran) for providing technical facilities, and to the participating families for their cooperation. Special appreciation is extended to Dr. Alireza Pourreza for his oversight of the experimental procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003es\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlireza Pourreza: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing\u0026mdash;original draft.\u003c/p\u003e\n\u003cp\u003eFarahzad Jabbari and Hamid Reza Kianifar: Patient recruitment, Clinical evaluation, Oral food challenge (OFC) and skin prick testing (SPT).\u003c/p\u003e\n\u003cp\u003eAbdolrahim Rezaee: Immunologic consultation, Writing\u0026mdash;review and editing.\u003c/p\u003e\n\u003cp\u003eFarjad Pourreza and Afsane Fadaee: Manuscript preparation and editing.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was received for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary files. De-identified diagnostic data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study (Ethics Code:\u0026nbsp;IR.MUMS.REC.86623) was reviewed and approved by the Ethics Committee of Mashhad University of Medical Sciences\u0026nbsp;(MUMS), Mashhad, Iran. Written informed consent was obtained from all participants\u0026rsquo; parents or legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnvari S, Miller J, Yeh CY, Davis CM. IgE-mediated food allergy. Clin Rev Allergy Immunol. 2019;57:244\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBardana EJ Jr.. Immunoglobulin E\u0026ndash; (IgE) and non\u0026ndash;IgE\u0026ndash;mediated reactions in the pathogenesis of atopic eczema/dermatitis syndrome (AEDS). Allergy. 2004;59(Suppl 78):25\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarni S, Liccioli G, Sarti L, Giovannini M, Novembre E, Mori F. Immunoglobulin E (IgE)-mediated food allergy in children: epidemiology, pathogenesis, diagnosis, prevention, and management. Med (Kaunas). 2020;56(3):111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Zhang N, Yang Y, Hong S, Bachert C. Local immunoglobulin E in nasal polyps: role and modulation. Front Immunol. 2022;13:961503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIweala OI, Burks AW. IgE producers in the gut expand the gut\u0026rsquo;s role in food allergy. Nat Rev Gastroenterol Hepatol. 2020;17(7):384\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDunnGalvin A, Dubois AE, Flokstra-de Blok BM, Hourihane JO. The effects of food allergy on quality of life. Food Allergy: Mol Basis Clin Pract. 2015;101:235\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacchia D, Melioli G, Pravettoni V, Nucera E, Piantanida M, Caminati M, et al. Guidelines for the use and interpretation of diagnostic methods in adult food allergy. Clin Mol Allergy. 2015;13:27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampson HA, Aceves S, Bock SA, James J, Jones S, Lang D, et al. Food allergy: a practice parameter update\u0026mdash;2014. J Allergy Clin Immunol. 2014;134(5):1016\u0026ndash;e104543.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol. 2001;107(5):891\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolmannskog S, Haneberg B. Immunoglobulin E in feces from children with allergy: evidence of local production of IgE in the gut. Int Arch Allergy Immunol. 1985;76(2):133\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalli SJ, Tsai M. IgE and mast cells in allergic disease. Nat Med. 2012;18(5):693\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurgel RK, Baroody FM, Damask CC, Mims JW, Ishman SL, Baker DP Jr, et al. Clinical practice guideline: immunotherapy for inhalant allergy. Otolaryngol Head Neck Surg. 2024;170(Suppl 1):S1\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNowak-Węgrzyn A, Katz Y, Mehr SS, Koletzko S. Non\u0026ndash;IgE-mediated gastrointestinal food allergy. J Allergy Clin Immunol. 2015;135(5):1114\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeehley T, Plunkett CH, Bao R, Choi Hong SM, Culleen E, Belda-Ferre P, et al. Healthy infants harbor intestinal bacteria that protect against food allergy. Nat Med. 2019;25(3):448\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMora JR, von Andrian UH. Differentiation and homing of IgA-secreting cells. Mucosal Immunol. 2008;1(2):96\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen KW, Marusciac L, Tamas PT, Valenta R, Panaitescu C. Ragweed pollen allergy: burden, characteristics, and management of an imported allergen source in Europe. Int Arch Allergy Immunol. 2018;176(3\u0026ndash;4):163\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViswanathan RK, Mathur SK. Role of allergen sensitization in older adults. Curr Allergy Asthma Rep. 2011;11:427\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerin MC, L\u0026oacute;pez-Exp\u0026oacute;sito I, Sampson HA, Nowak-Wegrzyn A. Detection of milk-specific IgE and IgA in stool samples from children with food allergy. J Allergy Clin Immunol. 2011;128(5 Suppl):S2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolmannskog S. Immunoglobulin E in feces of children with intestinal \u003cem\u003eAscaris lumbricoides\u003c/em\u003e infestation. Int Arch Allergy Immunol. 1986;80(4):417\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGevaert P, Holtappels G, Johansson SG, Cuvelier C, Van Cauwenberge P, Bachert C. Organization of secondary lymphoid tissue and local IgE formation to \u003cem\u003eStaphylococcus aureus\u003c/em\u003e enterotoxins in nasal polyp tissue. Allergy. 2005;60(1):71\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong J, Wang H, Zhang YN, Cao PP, Liao B, Wang ZZ, et al. Ectopic lymphoid tissues support local immunoglobulin production in patients with chronic rhinosinusitis with nasal polyps. J Allergy Clin Immunol. 2018;141(3):927\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGevaert P, Nouri-Aria KT, Wu H, Harper CE, Takhar P, Fear DJ, et al. Local receptor revision and class switching to IgE in chronic rhinosinusitis with nasal polyps. Allergy. 2013;68(1):55\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaba S, Kondo K, Toma-Hirano M, Kanaya K, Suzukawa K, Ushio M, et al. Local increase in IgE and class switch recombination to IgE in nasal polyps in chronic rhinosinusitis. Clin Exp Allergy. 2014;44(5):701\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida T, Usui A, Kusumi T, Inafuku S, Sugiyama T, Koide N, et al. A quantitative analysis of cedar pollen-specific immunoglobulins in nasal lavage supported the local production of specific IgE, not of specific IgG. Microbiol Immunol. 2005;49(6):529\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoh RA, Joshi SA, Lee JY, Martin BA, Varma S, Kwok S, et al. Origins and clonal convergence of gastrointestinal IgE⁺ B cells in human peanut allergy. Sci Immunol. 2020;5(45):eaay4209.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","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":"Food allergy, fecal IgE, mucosal immunity, diagnostic accuracy, oral food challenge","lastPublishedDoi":"10.21203/rs.3.rs-8829639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8829639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Conventional diagnostic tools for food allergy (FA), such as the skin prick test (SPT) and serum allergen-specific IgE (sIgE, formerly RAST), primarily detect systemic sensitization and may fail to capture localized mucosal immune responses. Measurement of fecal-specific IgE (f-sIgE) offers a noninvasive means to assess intestinal allergic inflammation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: To validate f-sIgE as a complementary biomarker for mucosal FA and examine its diagnostic accuracy compared with standard systemic tests and oral food challenge (OFC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Forty-one children with clinically suspected FA confirmed by OFC underwent SPT, serum sIgE (kU/L), and f-sIgE (kU/g) testing for ten common allergens (milk, egg, peanut, hazelnut, wheat, soy, fish, tree nuts, shellfish, and apple). Fecal samples were processed using a pH-adjusted, protease-inhibited buffer and analyzed by immunoblotting. Diagnostic performance metrics (sensitivity, specificity, correlation with OFC) were calculated using a predefined f-sIgE cut-off of 3.5 kU/g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Of 41 participants, 17 (41.5%) were positive for f-sIgE despite negative SPT results. Seven showed discordant allergen-specific patterns between serum and fecal IgE. One representative case exhibited markedly elevated fecal hazelnut IgE (\u0026gt;100 kU/g) with symptom resolution following maternal dietary elimination. f-sIgE correlated strongly with OFC outcomes (r = 0.91, p \u0026lt; 0.01), yielding 89% sensitivity and 92% specificity at the 3.5 kU/g threshold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: f-sIgE reflects local IgE production distinct from systemic sensitization and complements conventional tests for FA. These findings suggest f-sIgE as a promising noninvasive biomarker of mucosal allergic sensitization and may inform future development of standardized fecal IgE assays after multicenter validation.\u003c/p\u003e","manuscriptTitle":"Fecal-specific IgE as a Noninvasive and Complementary Biomarker for Mucosal Food Allergy: A Clinical Validation Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 07:00:33","doi":"10.21203/rs.3.rs-8829639/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":"6ec06f56-62f0-4756-b4a6-976435ebcfa9","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-17T00:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 07:00:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8829639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8829639","identity":"rs-8829639","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}