Related Prolamins Research Articles

Positive association of serum IgG4 levels with modified marsh classification system in children with gluten enteropathy: a single centre experience

Background: Celiac disease or gluten enteropathy is an immune-mediated systemic disorder elicited by gluten and related prolamines in genetically susceptible individuals and is characterized by the presence of gluten enteropathy, celiac disease-specific antibodies, HLA-DQ2/DQ8 haplotypes. IgG4-related diseases is an increasingly recognized immune-mediated condition in autoimmune disorders such as primary sclerosing cholangitis, autoimmune hepatitis, and autoimmune thyroiditis, characterized by tissue fibrosclerosis and infiltration by IgG4-positive plasma cells and increased serum IgG4 concentrations. Methods: A total of 33 children with newly diagnosed celiac disease and 31 control subjects were included in this study. All suspected celiac disease children underwent duodenal biopsy and were diagnosed based on Marsh grading. Serum IgG4 level estimations were performed using an enzyme-linked immune sorbet assay method with a cut-off of 135 mg/dl for diagnosis. Results: A significant positive association between serum IgG4 levels and Marsh classification was found, the higher the levels of IgG4 in serum, the higher the grade on Marsh staging. Mean serum IgG4 levels in Marsh 3a was 126.32 mg/dl, in 3b was 171.35 mg/dl and in 3c was 209.24 mg/dl (p value=0.004) Conclusions: With increasing serum IgG4 levels, increased severity of damage was seen on biopsy specimens based on higher Marsh grade. To the best of found knowledge, this is the first study to establish the relation between IgG4 and mucosal damage in children with celiac disease.

The Role of Intestinal Microbiota in Celiac Disease and Further Therapeutic Perspectives.

Celiac disease (CD) is an immune-mediated enteropathy caused by exposure to gluten and related prolamins in genetically susceptible individuals. It is a complex genetic disorder with multiple contributing genes. Linkage studies have identified several genomic regions that probably contain CD susceptibility genes. The most important genetic factors are HLA-DQ2 and DQ8. Several known environmental triggers promote the onset of CD at any age after gluten introduction in individuals with a genetic background, such as viral infections and intestinal dysbiosis. Recent publications have described the interference of the intestinal microbiome in gluten metabolism, modulation of local immune reactions, and in maintaining normal gut permeability. These results have promoted further lines of research on the benefit of probiotic administration to prevent disease onset or alleviate clinical symptoms along with a gluten-free diet (GFD). The relationship between gut microbiome changes and the onset of CD is incompletely understood, still being the subject of current research. This narrative review analyzes the interplay between environmental factors, intestinal microbiome alterations, and the course of CD. Furthermore, this review sets out to discuss if modulation of intestinal microflora with pre- and probiotics along with a GFD could represent a reliable therapeutic target for celiac patients.

The Role of Physicians' Factors in Underdiagnosis of Celiac Disease in the Eastern Province of Saudi Arabia.

Background Celiac disease is an autoimmune disorder triggered by gluten and related prolamines, which can cause a variety of symptoms and complications if left untreated. Despite being a common lifelong disorder, it often goes undiagnosed for a long time, leading to negative impacts on patients' health and quality of life. The diagnosis of celiac disease requires the presence of celiac-specific autoantibodies and distinctive histological changes in the small intestinal mucosa. Lack of disease knowledge among healthcare professionals and patients' adherence to gluten-free diets may contribute to diagnostic delays. Objectives This study aims to assess the reasons for celiac disease underdiagnosis and identify the functional deficiencies of healthcare professionals in the diagnosis and treatment of celiac disease, particularly in the Saudi population. Materials and methods A cross-sectional, questionnaire-based study was conducted among physicians in the Eastern Province of Saudi Arabia during the year 2023, between May and July. Participants were asked to complete an online self-administered questionnaire that included questions about their demographic characteristics, professional experience, and knowledge and attitudes toward celiac disease. The study recruited gastroenterologists, gastroenterology fellows, internal and family medicine specialists, residents, and general practitioners working in private or public health centers in various cities of the Eastern Province. Results The data were collected from 180 physicians who fulfilled the inclusion criteria of the study, with most participants aged under 30 years and predominantly male. Family medicine and general practitioners were the most represented specialties. While 49.4% of physicians knew that adult celiac disease was rare, only 19.4% frequently recommended celiac disease serology to their patients. In terms of risk, most physicians knew that adult celiac disease was a moderately severe and disabling disease, but only 24.4% thought that the cancer risk in patients with celiac disease was moderate. About 75.6% of physicians had an overall poor knowledge level regarding celiac disease, with gastroenterologists and internal medicine specialists demonstrating better knowledge compared to other specialties (P = 0.001). Conclusion The study found that a majority of physicians in the Eastern Province of Saudi Arabia had poor knowledge about celiac disease. This lack of knowledge could have implications for patient care, as it could lead to delays in diagnosis, inadequate treatment, and increased risk of complications.

Extraintestinal manifestation of Celiac disease: A case series

Celiac disease (CD) is a multisystem chronic inflammatory disorder with an autoimmune component in genetically susceptible individuals elicited by gluten and related prolamines. CD is becoming a common disorder arising at any age with a broad spectrum of symptoms ranging from intestinal to extraintestinal manifestations. Extraintestinal symptoms being more common after 2 years of life, can affect various systems and organs of the body. Knowledge of the extraintestinal manifestations of celiac disease is of utmost importance for the diagnosis of CD. Owing to the high risk of complications among the undiagnosed cases of CD, patients with extraintestinal manifestations should undergo appropriate diagnosis and treatment for CD.

The HLA-DQ8 transgenic mouse: A model to study the immune and cytotoxic responses to wheat gliadin.

A complete understanding of celiac disease (CD) pathogenesis has been hindered to date because of the lack of adequate in vivo models. Herein, we describe two in vivo approaches in HLA-DQ8-transgenic mice to study the intrinsic cytoxicity and immune features of wheat gliadin. By adopting the first method, we explored the mucosal architecture of the small intestine following the intra-gastric administration of wheat gliadin in mice treated with indomethacin, an inhibitor of cyclooxygenases. Mice showed a significant reduction of villus height, increased crypt depth and increased intraepithelial lymphocytes. The second approach involved the mucosal sensitization to gliadin via the intranasal route. This protocol induced a Th1/Th17 phenotype in mesenteric lymph nodes, as described in CD. In conclusion, these methods remain instrumental to analyze in vivo distinct biological features of wheat gliadin and related prolamins. Furthermore, the sensitization protocol could be exploited to test innovative strategies downregulating the gliadin-specific immunity.

PTPRK, an EGFR Phosphatase, Is Decreased in CeD Biopsies and Intestinal Organoids.

Celiac disease (CeD) is an immune-mediated enteropathy triggered in genetically susceptible (HLA-DQ2/8) individuals by a group of wheat proteins and related prolamins from cereals. The celiac intestine is characterized by an inversion of the differentiation/proliferation program of the enterocytes, with an increase in the proliferative compartment and crypt hyperplasia, which are the mechanisms that regulate the increased proliferation in CeD that arenot completely understood.The aim of this study is to understand the role of Protein Tyrosine Phosphatase Receptor Type K (PTPRK), a nodal phosphatase that regulates EGFR activation in the proliferation of the enterocytes from CeD biopsies and organoids. The levels of PTPRK were evaluated by RT PCR, western blot (WB) and immunofluorescence techniques in intestinal biopsies and organoids from CeD patients and controls. Additionally, pEGFR and pERK were evaluated by WB and proliferation by BrdU incorporation. PTPRK si-RNA was silenced in CTR organoids and was overexpressed in CeD organoids. PTPRK was reduced in Gluten Containing Diet-Celiac Disease (GCD-CeD) and Potential-Celiac Disease(Pot-CeD) biopsies (p < 0.01-p < 0.05) whereas pEGFR (p < 0.01 p < 0.01), pERK (p < 0.01 p < 0.01) and proliferation were increased. (p < 0.05 p < 0.05) respect to the controls.The CeD organoids reproduced these same alterations. Silencing of PTPRK in CTR organoids increased pEGFR, pERK and proliferation. The overexpression of PTPRK in CeD organoids reduced pEGFR, pERK and proliferation. modulation of PTPRK levels can reduce or increase pEGFR, pERK and proliferation in CeD or CTR organoids, respectively. The CeD organoids can be a good model to study the mechanisms of the disease.

Fast blue B functionalized silica-polymer composite to evaluate 3,5-dihydroxyhydrocinnamic acid as biomarker of gluten intake

Celiac disease is an immune-mediated systemic disorder elicited by gluten and related prolamines in genetically susceptible individuals. The current treatment is a strict and lifelong gluten-free diet. However, compliance with the gluten-free diet is not always adequate and many food products contain low concentrations of gluten. The determination of dietary transgressions is a challenge for patients, physicians and dietitians. Alkylresorcinols (AR) have been proposed as sensitive and specific biomarkers of gluten consumption. In this work silica–polymer composites doped with fast blue B reagent (FB) have been used to estimate alkylresorcinols in biological samples. The proposed colorimetric device was synthetized by immobilizing FB into polydimethylsiloxane-tetraethylortosilicate composite. The assay was based on the spontaneous release of FB to the solution containing AR (3,5-dihydroxyhydrocinnamic acid, DHCA) and the azocomplexe formation (520 nm). The response was evaluated with UV-vis spectroscopy and chromatography (in-tube SPME-Capillary LC-DAD) to isolate DHCA signal. The spectroscopic analysis can be used as a screening tool to differentiate positive and negative samples. Meanwhile, the chromatographic assay was necessary to isolate DHCA response in positive samples. LOD was 60 ng mL -1 by adding a SPE step. Precision provided adequate results (RSD < 15%). Preliminary studies in urine samples displayed successful results, showing DHCA can be used as biomarker of gluten intake. The here proposed methodology clearly simplifies the dietary transgression evaluation thanks to the development of a pre-screening tool before the chromatographic analysis.

Coeliac disease: making the diagnosis

### Key points Coeliac disease (CD) is a systemic, immune-mediated disorder precipitated by gluten and related prolamins. It is characterised by gut enteropathy. It can manifest at any age after...

Pilot scale production of a non-immunogenic soluble gluten by wheat flour transamidation with applications in food processing for celiac-susceptible people

Celiac disease (CD) is an immune-mediated disease triggered by wheat gluten and related prolamins. A lifelong gluten-free (GF) diet is mandatory to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase of gluten was effective in suppressing the gliadin-specific inflammatory response in CD patients without influencing the main technological properties of wheat flour or semolina. In this study, we produced on a pilot scale a soluble form of transamidated gluten (soluble protein fraction, spf), characterised by a high protein content (88 mg/ml), while native gluten was dramatically reduced (32 ± 2 ppm; R5-ELISA). Using HLA-DQ8 transgenic mice as a CD model, we found suppression of interferon-γ secretion in gliadin-specific CD4+ T cells challenged with spf-primed dendritic cells. In terms of functional properties, spf showed both solubility and emulsifying activity values within the range of commercial soluble glutens. Notably, dough prepared by mixing rice flour with spf could leaven. After baking, blended rice bread had a higher specific volume (2.9 ± 0.1) than control rice bread (2.0 ± 0.1) and acquired wheat-like sensory features. Taken together, our results highlighted the technological value of transamidated soluble gluten to improve both nutritional and sensory parameters of GF food.

Brain Opioid Activity and Oxidative Injury: Different Molecular Scenarios Connecting Celiac Disease and Autistic Spectrum Disorder.

Celiac Disease (CD) is an immune-mediated disease triggered by the ingestion of wheat gliadin and related prolamins from other cereals, such as barley and rye. Immunity against these cereal-derived proteins is mediated by pro-inflammatory cytokines produced by both innate and adaptive system response in individuals unable to adequately digest them. Peptides generated in this condition are absorbed across the gut barrier, which in these patients is characterized by the deregulation of its permeability. Here, we discuss a possible correlation between CD and Autistic Spectrum Disorder (ASD) pathogenesis. ASD can be induced by an excessive and inappropriate brain opioid activity during the neonatal period. Cereal-derived peptides produced in celiac patients cross the blood–brain barrier and bind to endogenous opioid receptors interfering with neurotransmission and generating deleterious effects on brain maturation, learning and social relations. Moreover, an increase in oxidative stress and a decrease in the antioxidant capacity, as well as an extended mitochondrial impairment in the brain, could represent a possible connection between ASD and CD. Therefore, we critically discuss the proposed relationship between ASD and CD and the possible usefulness of a gluten-free diet in ASD patients.

ASSESSMENT OF CLINICAL PROFILE OF CELIAC DISEASE AMONG CHILDREN AT TERTIARY CARE HOSPITAL

Background: Celiac disease is reported to be the most common genetic and food related intolerance, globally. Celiac disease is an autoimmune disease that reported among genetically susceptible individuals. The autoimmune process is reported to be triggered by a well-identified associated environmental factor known as gluten and its related prolamins which are present in wheat, barley and rye.&#x0D; Material &amp; Methods: The present prospective study was conducted at department of pediatrics of our tertiary care hospital. The study was an observational study on 30 self-reported patients during a period of one year from January 2017 to December 2017. Patients without any exclusion criteria of sex, religion and socioeconomic status were enrolled by simple random sampling.&#x0D; Results: In the present study, on the basis of clinical symptoms we found that in the age group of less than 5 years 75% had diarrhea, 87.5% had abdominal distension, 87.5% had abdominal pain, 75% had anorexia. In the age group of 5-10 years 50% had diarrhea, 66.6% had abdominal distension, 58.3% had abdominal pain, 66.6% had anorexia and 16.6% had constipation. In the age group of more than 10 years 70% had diarrhea, 60% had abdominal distension, 50% had abdominal pain, 40% had anorexia and 20% had constipation.&#x0D; Conclusion: We concluded from the present study that most common clinical symptom among children was diarrhea followed by abdominal distention, pain in abdomen, anorexia, constipation, pallor,edema, vitamin deficiencies and mouth ulcers.&#x0D; Keywords: Celiac disease, IgA-anti tissue transglutaminase antibodies, Screening.

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oat Products by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action 2018.15

Abstract Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples &amp;gt;10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.

Quantification of Wheat, Rye, and Barley Gluten in Oat and Oat Products by ELISA RIDASCREEN® Total Gluten: Collaborative Study, First Action 2018.15.

Background: Since its introduction to the analytical community, the R5 method to quantify gluten led to a strong improvement of the situation for the food industry and celiac patients. During recent years, some questions arose on the use of the Codex Alimentarius factor of two to convert from prolamins to gluten, an overestimation of rye and barley, inadequate detection of glutelins, and the inhomogeneous distribution of gluten in oats. These limitations of the R5 method, especially when measuring oat samples, led to AOAC Standard Method Performance Requirement (SMPR®) 2017.021, which was approved by stakeholders in 2017. Objective: We present a collaborative study of a method for the quantitative analysis of wheat, rye, and barley gluten in oat and oat products using a sandwich ELISA that is based on four different monoclonal antibodies including the R5 monoclonal anitbody. Methods: The sandwich ELISA detects intact gliadins and related prolamins from rye and barley, high-molecular-weight (HMW) glutenin subunits (GS) from wheat, HMW secalins from rye, and low-molecular-weight (LMW) GS from wheat. It does not detect D-hordeins from barley. Samples are extracted by Cocktail solution, subsequently followed by 80% ethanol, and analyzed within 50 min. Results: The measurement range is between 5 and 80 mg/kg gluten using a calibrator made out of a gluten extract from four different wheat cultivars. The results of the collaborative test with 19 participating laboratories showed recoveries ranging from 99 to 137% for all three grain sources. Relative reproducibility SDs for samples >10 mg/kg gluten ranged from 10 to 53%. Conclusions: The collaborative study results confirmed that the method is accurate and suitable to measure gluten from all three grain sources and has demonstrated performance on oat matrices, which meets the criteria as specified in SMPR 2017.021. Data from in-house validation experiments are available as Annex B to this publication.

Influence of HLA on clinical and analytical features of pediatric celiac disease

BackgroundCeliac disease (CD) is triggered by gluten and related prolamines in genetically susceptible individuals. We aimed to investigate the influence of HLA-DQ genotypes in clinical, serological and histological features related to CD.MethodsA retrospective observational study was performed including 463 Spanish patients with biopsy-proven CD. Clinical, serological, histological and HLA-DQ genetic data were collected from each participant. The presence of a family history of CD was also considered. Bivariate (chi-square tests or the Fisher’s exact test) and multivariate (logistic regression after adjusting for age and sex) analyses were performed to assess the association between clinical and laboratory parameters with HLA-DQ.ResultsA predominance of females (62%), classical clinical presentation (86%) and positive anti-transglutaminase 2/endomysium antibodies (99%) was observed in our sample, with a mean age at onset of 2.6 ± 0.1 years. Five percent of our patients were first-degree relatives of subjects with CD, with HLA-DQ genetics showing increased homozygosity of HLA-DQ2.5 (p = 0.03) and HLA-DQ8 (p = 0.09). In the non-CD family history group, an association between delayed disease onset and HLA-DQ8 carriage was observed (p < 0.001), besides an influence of HLA-DQB1*02 gene dosage on clinical presentation and severity of histological damage (after adjusting for age and sex, p = 0.05 and p = 0.02, respectively) and a trend towards presence of specific antibodies (p = 0.09). These associations could not be evaluated properly in the group of patients with affected first-degree relatives due to the small sample size.ConclusionsHLA-DQ genotypic frequencies differ slightly between CD patients depending on their family history of CD. In patients lacking CD first-degree relatives, carriage of HLA-DQ2.5 with double dose of HLA-DQB1*02 seems to be associated with classical clinical presentation and more severe histological damage.

Extra-Intestinal Manifestations of Coeliac Disease in Children: Clinical Features and Mechanisms.

Celiac disease (CD) is a systemic autoimmune disease due to a dysregulated mucosal immune response to gluten and related prolamines in genetically predisposed individuals. It is a common disorder affecting ~1% of the general population, its incidence is steadily increasing. Changes in the clinical presentation have become evident since the 80s with the recognition of extra-intestinal symptoms like short stature, iron deficiency anemia, altered bone metabolism, elevation of liver enzymes, neurological problems. Recent studies have shown that the overall prevalence of extra-intestinal manifestations is similar between pediatric and adult population; however, the prevalence of specific manifestations and rate of improvement differ in the two age groups. For instance, clinical response in children occurs much faster than in adults. Moreover, an early diagnosis is decisive for a better prognosis. The pathogenesis of extra-intestinal manifestations has not been fully elucidated yet. Two main mechanisms have been advanced: the first related to the malabsorption consequent to mucosal damage, the latter associated with a sustained autoimmune response. Importantly, since extra-intestinal manifestations dominate the clinical presentation of over half of patients, a careful case-finding strategy, together with a more liberal use of serological tools, is crucial to improve the detection rate of CD.

Oral Manifestations of Celiac Disease and Wheat Allergy: Report of Three Cases and Review of Literature

Celiac Disease (CD) is an immune-mediated systemic disease, caused by gluten and related prolamins, in genetically susceptible individuals. It is characterized by gluten-dependent clinical manifestations, CD-specific antibodies, HLA DQ2 or DQ8 haplotypes, and enteropathy. Most patients are asymptomatic or with mild manifestations. It mainly affects the gastrointestinal system causing symptoms and signs such as diarrhea, recurrent abdominal pain and abdominal distension; also extradigestive manifestations. Patients with CD may have oral manifestations that include recurrent aphthous stomatitis (RAS), enamel hypoplasia, atrophic glossitis, among others. The diagnosis of CD is based on clinical history, serology, endoscopy and histology. There are other entities that should be differentiated from CD, such as non-celiac gluten sensitivity and wheat allergy. The aim of this study is to present three patients with a history of RAS and other clinical manifestations associated with the early diagnosis of CD and food allergy.

Cardiometabolic risk factors in children with celiac disease on a gluten-free diet.

Celiac disease (CD) is an immune-mediated systemic condition evoked by gluten and related prolamines in genetically predisposed subjects. It is characterised by a variable combination of gluten-dependent clinical symptoms, CD-specific antibodies, HLA-DQ2 and HLA-DQ8 haplotypes, and enteropathy. The only therapy of CD consists of a life-long gluten free diet (GFD). Strict GFD adherence results in full clinical, serological and histological remission, avoiding long-term complications in CD patients. However, this diet is not without problems. Gluten free products have high levels of lipids, sugar and salt to improve food palatability and consistency, and subjects with CD show an excessive consumption of hypercaloric and hyperlipidic foods to compensate dietetic restriction. GFD may therefore have a negative impact on cardiometabolic risk factors such as obesity, serum lipid levels, insulin resistance, metabolic syndrome, and atherosclerosis. In adults, some studies have suggested that GFD have a beneficial effect on cardiovascular profile, whereas others have shown an atherogenic effect of GFD. In children, very few studies are available on the issue. Thus, the aim of the present narrative review was to analyze the current clinical evidence on the impact of GFD on cardiometabolic risk factors in children with CD.

Epidemiology of Celiac Disease in Slovakia: Life Conditions of Celiac Disease Patients in Slovakia

Celiac disease (CD) is a chronic autoimmune disorder, elicited by gluten and related prolamines, with gluten dependant clinical manifestation in genetically susceptible individuals, with specific antibodies, HLA haplotyps and enteropathy. In Slovakia epidemiology situation of patients with diagnosed CD was being repeatedly surveyed. Patient with CD in Slovakia has possibility for prescriptions up to 50 specific gluten free products, partially covered by health insurance.

Degree of Mucosal Damage and Clinical Presentation of Celiac Disease – Is There a Connection?

Celiac disease (CD) is an immune-mediated enteropathy induced by gliadin and related prolamins. This study was designed to evaluate the correlation between the degree and uniformity of mucosal damage and forms of CD. The study included a total of 85 children (33 boys and 52 girls, mean age 6.59 years) hospitalized due to CD. Patients were assigned to one of three groups based on their form of disease manifestation: (1) patients with classical form; (2) patients with atypical form; and (3) patients with asymptomatic celiac disease. The diagnosis of CD was based on positive CD specific antibodies, HLA typing and histological analysis of multiple biopsy samples. Histological changes were classified using modified Marsh criteria. Forty one patients had classical form, 32 had atypical form, while 12 patients had asymptomatic form of celiac disease. There was no difference in the degree of damage on small bowel samples between different clinical forms of celiac disease (p=0.079). The frequency of uniformity of enteropathy does not depend on clinical form of the disease (p=0.882). Uniform small bowel mucosal damage was more common in cases of severe degree of mucosal damage (p=0.017). The degree of damage and uniformity of changes in duodenal biopsy specimens did not correlate with mode of presentation, while uniform mucosal damage increased with severity of enteropathy.

Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease.

BackgroundCeliac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities.ResultsGluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a prokaryotic expression vector, which provides a C-terminal hexahistidine tag.ScFvs from several bacterial clones were expressed in soluble form and crude cell lysates screened for binding to PT-Gliadin by ELISA. We identified an enriched scFv motif, which showed reactivity to PT-Gliadin. One selected scFv candidate was expressed and purified to homogeneity. Polyclonal anti-PT-Gliadin IgY, purified from egg yolk of immunized chicken, served as control. ScFv binds in a dose-dependent manner to PT-Gliadin, comparable to IgY. Furthermore, IgY competitively displaces scFv from PT-Gliadin and natural wheat flour digest, indicating a common epitope of scFv and IgY. ScFv was tested for reactivity to different gastric digested dietary grain flours. ScFv detects common and khorasan wheat comparably with binding affinities in the high nanomolar range, while rye is detected to a lesser extent. Notably, barley and cereals which are part of the gluten-free diet, like corn and rice, are not detected by scFv. Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv. This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.ConclusionScFv can be of benefit for future CD treatment regimes.