Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing.

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients' mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.

Exploring Gluten Assessment in Marketed Products through a Sandwich ELISA Methodology Based on Novel Recombinant Antibodies.

This study presents the development of a sandwich ELISA method for gluten detection in foods, using recombinant Fab antibody fragments against gliadin. The Fabs were chemically biotinylated and immobilized on streptavidin-coated plates as capture antibodies, while alkaline phosphatase-conjugated Fabs were used as detection antibodies. Four different gliadin-binding Fabs were tested and the Fab pair Fab8E-4 and Fab-C showed the best compatibility. An indirect sandwich immunoassay, using unmodified Fab8E-4 for capture and Fab-C as the detection antibody, achieved a detection limit of 26 ng/mL of gliadin, corresponding to 10 mg/kg of gluten in foods. No cross-reactivity was observed against 60 gluten-free species commonly used in the food industry. Analysis of 50 commercial products demonstrated consistent results compared to the standard method for gluten detection. The complete lack of cross-reactivity of the developed immunoassay with oat products potentially provides an advantage over other gluten detection systems.

Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen's Egg Detection.

Hen's egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen's ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.

Production and Characterization of Novel Fabs Generated from Different Phage Display Libraries as Probes for Immunoassays for Gluten Detection in Food.

Gluten is the main fraction of wheat proteins. It is widely used in the food industry because of the properties that are generated in the dough, but it is also able to trigger diseases like allergies, autoimmunity processes (such as celiac disease), and intolerances in sensitized persons. The most effective therapy for these diseases is the total avoidance of gluten in the diet because it not only prevents damage but also enhances tissue healing. To ensure the absence of gluten in food products labeled as gluten-free, accurate detection systems, like immunoassays, are required. In this work, four recombinant Fab antibody fragments, selected by phage display technology, were produced and tested for specificity and accuracy against gluten in experimental flour mixtures and commercial food products. A high-affinity probe (Fab-C) was identified and characterized. An indirect ELISA test was developed based on Fab-C that complied with the legal detection limits and could be applied in the assessment of gluten-free diets.

Development of a new recombinant antibody, selected by phage-display technology from a celiac patient library, for detection of gluten in foods.

Gluten, a group of ethanol-soluble proteins present in the endosperm of cereals, is extensively used in the food industry due to its ability to improve dough properties. However, gluten is also associated with a range of gluten-related diseases (GRDs), such as wheat allergies, celiac disease, and gluten intolerance. The recommended treatment for GRDs patients is a gluten-free diet. To monitor adherence to this diet, it is necessary to develop gluten-detection systems in food products. Among the available methods, immunodetection systems are the most popular due to their simplicity, reproducibility, and accuracy. The aim of this study was to generate novel high-affinity antibodies against gluten to be used as the primary reactant in an enzyme-linked immunosorbent assay (ELISA) test. These antibodies were developed by constructing an immune library from mRNA obtained from two celiac patients with a high humoral response to gluten-related proteins. The resulting library (composed by 1.1x107) was subjected to selection against gliadin using phage display technology. Following several rounds of selection, the Fab-C was selected, and demonstrated good functionality in ELISA tests, presenting a limit of detection of 15 mg/kg for detection of gluten in spiked mixtures and food products. The methodology can discriminate gluten-free products according to the current legislation.

Construction of a Fab Library Merging Chains from Semisynthetic and Immune Origin, Suitable for Developing New Tools for Gluten Immunodetection in Food.

The observed increase in the prevalence of gluten-related disorders has prompted the development of novel immunological systems for gluten detection in foodstuff. The innovation on these methods relies on the generation of new antibodies, which might alternatively be obtained by molecular evolution methods such as phage display. This work presents a novel approach for the generation of a Fab library by merging semi-synthetic heavy chains built-up from a pre-existent recombinant antibody fragment (dAb8E) with an immune light chain set derived from celiac donors. From the initial phage population (107 candidates) and after three rounds of selection and amplification, four different clones were isolated for further characterization. The phage Fab8E-4 presented the best features to be applied in an indirect ELISA for the detection of gluten in foods, resulting in improved specificity and sensitivity.

Production of a Recombinant Single-Domain Antibody for Gluten Detection in Foods Using the Pichia pastoris Expression System.

The detection of gluten in foodstuffs has become a growing concern in food allergen management as a result of the high ratio of population sensitive to the main gluten-containing cereals. In this study, a promising single-domain antibody previously isolated by phage display (dAb8E) was produced in Pichia pastoris resulting in high levels of the antibody fragment expression (330 mg/L). The purified dAb8E was proved to specifically bind to gluten proteins from wheat, barley and rye, exhibiting no cross reaction to other heterologous species. The dynamic range of the sandwich enzyme-linked immunosorbent assay (ELISA) covered 0.1 to 10 µg/mL of gliadin, reaching a limit of detection of 0.12 µg/mL. When experimental binary mixtures of the target cereals were analyzed, the limit of detection was 0.13 mg/g, which would theoretically correspond to gluten concentrations of approximately 13 mg/kg. Finally, thirty commercially available food products were analyzed by means of the developed assay to further confirm the applicability of the dAb8E for gluten determination. The proposed methodology enabled the generation of a new gluten-specific nanobody which could be used to guarantee the appropriate labelling of gluten-free foods.

From Polyclonal Sera to Recombinant Antibodies: A Review of Immunological Detection of Gluten in Foodstuff.

Gluten is the ethanol-soluble protein fraction of cereal endosperms like wheat, rye, and barley. It is widely used in the food industry because of the physical-chemical properties it gives to dough. Nevertheless, there are some gluten-related diseases that are presenting increasing prevalences, e.g., celiac disease, for which a strict gluten-free diet is the best treatment. Due to this situation, gluten labeling legislation has been developed in several countries around the world. This article reviews the gluten immune detection systems that have been applied to comply with such regulations. These systems have followed the development of antibody biotechnology, which comprise three major methodologies: polyclonal antibodies, monoclonal antibodies (mAbs) derived from hybridoma cells (some examples are 401.21, R5, G12, and α-20 antibodies), and the most recent methodology of recombinant antibodies. Initially, the main objective was the consecution of new high-affinity antibodies, resulting in low detection and quantification limits that are mainly achieved with the R5 mAb (the gold standard for gluten detection). Increasing knowledge about the causes of gluten-related diseases has increased the complexity of research in this field, with current efforts not only focusing on the development of more specific and sensitive systems for gluten but also the detection of protein motifs related to pathogenicity. New tools based on recombinant antibodies will provide adequate safety and traceability methodologies to meet the increasing market demand for gluten-free products.