A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins

Osman, Awad A.; Uhlig, Holm H.; Valdés, Israel; Amin, Maha M; Méndez, Enrique; Mothes, Thomas

doi:10.1097/00042737-200110000-00011

Cited by 131 publications

(82 citation statements)

References 26 publications

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“…The response of Gli 1 aptamer to the recognition epitope of the official R5 method, that is, the pentapeptide QQPFP [23] was also tested. This specific sequence is not present in the immunodominat 33-mer peptide used for aptamer selection but it is present in wheat, rye and barley prolamins.…”

Section: Figurementioning

confidence: 99%

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Amaya-González

de‐los‐Santos‐Álvarez

Miranda‐Ordieres

et al. 2015

Anal Bioanal Chem

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Enzyme-immune assays are currently the methods of choice for gluten control in foods labelled as "gluten free", providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardized method of analysis. We explore new methods for quantitatively determine gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilized onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kind of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods.

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Section: Figurementioning

confidence: 99%

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Amaya-González

de‐los‐Santos‐Álvarez

Miranda‐Ordieres

et al. 2015

Anal Bioanal Chem

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“…Although R5 antibody is declared to have no cross-reactivity with oat proteins, avenins may contain QQQPF sequences and they can also react properly with R5 antibody (Comino et al, 2011;Ellis et al, 1998;Osman et al, 2001). However, some doubts can occur in case of the extrapolation of the results obtained with gliadin-detecting antibodies in order to measure the toxicity of other protein species.…”

Section: Resultsmentioning

confidence: 99%

Oat raw materials and bakery products – amino acid composition and celiac immunoreactivity

Mickowska

Litwinek

Gambuś

2016

Acta Sci Pol Technol Aliment

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Background. The aim of this study was to compare the biochemical and immunochemical properties of avenins in some special oat raw materials and additionally the possibility of using them as a raw material for the gluten-free bakery products. Materials and methods. The compared oat raw materials were -oat fl akes, commercial oat fl ours (including gluten-free oat fl our) and residual oat fl our, which is by-product of β-glucan preparation. Biochemical characteristic included amino acid compositions and SDS-PAGE profi les of extracted avenins. The immunochemical reactivity with polyclonal anti-gluten and monoclonal anti-gliadin antibodies was evaluated qualitatively and quantitatively by immunoblotting and ELISA methods. Additionally, experimental bakery products made of examined raw materials were assessed according to their suitability for the celiac patients' diet. Results. The highest protein content was measured in the β-glucan preparation "Betaven" and gluten-free oat fl our. Proteins of all materials are rich in glutamic and aspartic acid, leucine and arginine. Proportions of amino acids in avenins extracted from most of oat raw materials are similar, excluding gluten-free oat fl our, which has a very low avenin content and proportions of individual amino acids are diff erent. The SDS-PAGE protein pattern consisted of proteins with molecular weight of about 25-35 kDa. Polyclonal anti-gluten antibody recognized all protein fractions of molecular weight higher than 20 kDa. Quantitative ELISA analysis shows that the majority of samples has a gliadin-like protein content within the range of 80-260 mg/kg, excluding gluten-free fl ours and corresponding bakery products. Altogether, β-glucan preparation has extremely high level of gliadin-like proteins. Conclusion. In the examined oat raw materials and foods the contents of immunoreactive amino acid sequences exceeded the limit of 20 mg/kg (considered as gluten-free) except for gluten-free fl ours (oat and the prepared mixture) and the bakery products based on gluten-free fl ours. Unfortunately, the rest of oat raw materials and products cannot be considered gluten-free.

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“…PS from all subjects showed similar patterns of weak reactivity in this region, and no group-specific differences were noted (data not shown). Recognition of salivary proteins was also investigated with a second antibody, R5, directed at the pentapeptide QQPFP and related sequences comprised in immunogenic gluten epitopes (25,36,40,57 produced by PBMCs were analyzed after 24-h incubation to the respective samples. PT-TG2-treated H2O alone served as the negative control.…”

Section: Salivary Prps Show Sequence Similarity To Gliadin Immunogenimentioning

confidence: 99%

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease

Tian

Leffler

Kelly

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Helmerhorst EJ. Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease. Am J Physiol Gastrointest Liver Physiol 309: G910 -G917, 2015. First published October 1, 2015; doi:10.1152/ajpgi.00157.2015.-Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immunemediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to prolinerich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten. celiac disease; immune response; salivary protein; gluten; mouse model CELIAC DISEASE (CD) is a T cell-mediated inflammatory enteropathy that manifests itself in genetically predisposed individuals. In its etiology, both genetic and environmental factors are implicated. The major genetic risk factors are carriage of HLA-DQ2 heterodimers, expressed in 90 -95% of CD patients, or HLA-DQ8 heterodimers, expressed in the remainder of CD patients. The primary environmental cause of CD is ingested gluten, a heterogeneous mixture of glutamine-and proline-rich storage proteins present in wheat, rye, and barley (10,12,22,46). According to solubility in alcoholic solutions, gluten is divided into soluble gliadins and insoluble glutenins. Gluten proteins are unusual because they have a high content of proline (P) and glutamine (Q). In the most abundant gliadins, Q comprises 35-38% of the total amino acids, while P ranges from 13-17% (49, 62, 63). The high P conten...

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A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins

Abstract: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.

Cited by 131 publications

References 26 publications

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Oat raw materials and bakery products – amino acid composition and celiac immunoreactivity

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease

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A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins

Abstract: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.

Cited by 131 publications

References 26 publications

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Sensitive gluten determination in gluten-free foods by an electrochemical aptamer-based assay

Oat raw materials and bakery products &#8211; amino acid composition and celiac immunoreactivity

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease

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Oat raw materials and bakery products – amino acid composition and celiac immunoreactivity