Celiac disease-specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity

Identification of key effects causing weak performance of allergen analysis in processed food matrices

The weaker performance of generally used analytical methods for allergen analysis in processed foods can be connected to protein denaturation. To understand the nature of protein denaturation processes, experimental but realistic model matrices (corn starch based mixture, hydrated dough, and heat treated cookies) were developed that contain a defined amount of milk, egg, soy, and wheat proteins individually or in combination. The protein subunit composition was investigated in every processing phase, i.e. after mixing, dough formation, and baking. SDS-PAGE measurements were carried out to monitor the protein distribution of sample food matrices in non-reducing and reducing gels. The results clearly show that the highly decreased protein solubility is caused by denaturation, aggregation, or complex formation, which are the most significant factors in poorer analytical performances. Solubility can only partly be improved with the application of reducing agents or surfactants, and the rate of improvement is depending on the proteins and the matrices

Investigation of the effects of sample preparation on gluten quantitation in rye and barley flours

Proper gluten quantitation is essential for providing safe gluten-free food for patients living with celiac disease (CD). However, gluten quantitation faces several challenges: the lack of a reference method and certified reference materials, the variability of methods and the effects of genetic and environmental factors on gluten. Among all these challenges our research group focuses on gluten reference material development. Gluten content is determined by enzyme linked immunosorbent assay (ELISA) methods to obtain comparable data for the selection of cultivars used in our reference material development efforts. As ELISA methods are developed for determining low gluten concentrations, application for these special research purposes requires a 10,000-fold dilution. The formerly performed process was a post-extraction liquid dilution that proved to be sufficient for wheat samples. However, gluten contents of rye and barley samples were found to be overestimated by ELISA methods. One of the suggested reasons is the structural and solubility changes of gluten proteins during the dilution process. Therefore, our present study focuses on the comparison of the original dilution method and a revised version using solid-phase dilution in a gluten-free matrix

Applicability of ELISA methods for high gluten-containing samples

Quantitation of gluten in gluten-free products is a great challenge as it is hindered by several factors including the lack of certified reference materials. To resolve this problem, our research group, in cooperation with other international experts, started a series of experiments with the goal of the production of a suitable gluten reference material. As a part of this research, several different wheat cultivars and their isolated gluten proteins were characterized by different methods, including enzyme-linked immunosorbent assay (ELISA). However, we need to know the performance of the ELISA methods used for this special area of research. During the present work we investigated the accuracy and precision of two different ELISA methods for our own laboratory conditions and special sample matrices (wheat flours and gliadin isolate). We have found that the tested performance characteristics of the methods seem to be appropriate on a case-by-case basis, but the long-term measurement uncertainty is higher, which makes it difficult to evaluate the results obtained with the ELISA method for these types of samples

Investigation of the effects of food processing and matrix components on the analytical results of ELISA using an incurred gliadin reference material candidate

Disorders induced by cereal proteins (e.g. wheat allergy, celiac disease) are widespread in human population. Since their only effective treatment is the avoidance of the problematic proteins, patients have to be familiar with the composition of food products. For checking special foods produced for them, proper analytical methods are necessary. At the moment, in gluten analysis there are no reference methods and reference materials which model real food matrices. During the production and experimental utilisation of our previously developed reference material candidate, numerous questions emerged. As our model product is a real food matrix, interactions can be present between gluten proteins and other macro and micro components. Fat content of the baked cookies is almost 20%, which might affect the results of ELISA measurements. The detectable gluten content is significantly increasing after the defatting procedure, as a pre-treatment of samples. Moreover, baking is a common food processing step that might modify the structure of gluten proteins leading to denaturation and aggregation. In the soluble protein fraction the amount of low molecular weight proteins increases, while that of high molecular weight proteins decreases during the baking procedure

ELISA response and gliadin composition of different wheat cultivars grown in multiple harvest years

In special dietary products for people intolerant to gluten, gluten content is not supposed to exceed the regulatory thresholds. Enzyme-linked immunosorbent assays (ELISAs) are routinely used to quantitate gluten in these products. They measure gliadin/gluten with high specificity and sensitivity, but they have some limitations. Quantitative and qualitative variability of the target proteins among wheat cultivars is a factor that may cause inaccurate results. One of the aims of this work was to characterize the protein composition of five wheat cultivars grown in multiple harvest years and their blends by reversed-phase high-performance liquid chromatography (RP-HPLC). The gliadin/gluten content of these wheat flours was also analysed with two commercial ELISA kits. The effect of differences in protein profiles between the flours from an individual cultivar and the blend of five cultivars, harvest years, as well as processing procedures (dough forming and baking) on the results of two ELISA kits was investigated and their relative magnitude was determined. Among the factors investigated, the differences between flours had the greatest impact on gliadin recoveries

Investigation of the effects of sample preparation on gluten quantitation in rye and barley flours

AbstractProper gluten quantitation is essential for providing safe gluten-free food for patients living with celiac disease (CD). However, gluten quantitation faces several challenges: the lack of a reference method and certified reference materials, the variability of methods and the effects of genetic and environmental factors on gluten. Among all these challenges our research group focuses on gluten reference material development. Gluten content is determined by enzyme linked immunosorbent assay (ELISA) methods to obtain comparable data for the selection of cultivars used in our reference material development efforts. As ELISA methods are developed for determining low gluten concentrations, application for these special research purposes requires a 10,000-fold dilution. The formerly performed process was a post-extraction liquid dilution that proved to be sufficient for wheat samples. However, gluten contents of rye and barley samples were found to be overestimated by ELISA methods. One of the suggested reasons is the structural and solubility changes of gluten proteins during the dilution process. Therefore, our present study focuses on the comparison of the original dilution method and a revised version using solid-phase dilution in a gluten-free matrix.</jats:p