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.

BackgroundCeliac disease (CD) is one of the most common food-related chronic disorders. It is mediated by the dietary consumption of prolamins, which are storage proteins of different grains. So far, no therapy exists and patients are bound to maintain a lifelong diet to avoid symptoms and long-term complications. To support those patients we developed a tandem single chain Fragment variable (tscFv) acting as a neutralizing agent against prolamins. We recombinantly produced this molecule in E. coli, but mainly obtained misfolded product aggregates, so-called inclusion bodies, independent of the cultivation strategy we applied.ResultsIn this study, we introduce this novel tscFv against CD and present our strategy of obtaining active product from inclusion bodies. The refolded tscFv shows binding capabilities towards all tested CD-triggering grains. Compared to a standard polyclonal anti-PT-gliadin-IgY, the tscFv displays a slightly reduced affinity towards digested gliadin, but an additional affinity towards prolamins of barley.ConclusionThe high binding specificity of tscFv towards prolamin-containing grains makes this novel molecule a valuable candidate to support patients suffering from CD in the future.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0443-0) contains supplementary material, which is available to authorized users.

Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell-substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.

Spider venoms are highly complex mixtures. Numerous spider venom metabolites are uniquely found in spider venoms and are of interest concerning their potential use in pharmacology, agriculture, and cosmetics. A nontargeted ultra-high performance high-resolution electrospray tandem mass spectrometry (UHPLC-HR-ESI-MS/MS) approach offers a resource-saving way for the analysis of crude spider venom. However, the identification of known as well as the structure elucidation of unknown low molecular mass spider venom compounds based on their MS/MS spectra is challenging because (1) acylpolyamine toxins are exclusively found in spider and wasp venom, (2) reference MS/MS spectra are missing in established mass spectrometry databases, and (3) trivial names for the various toxin metabolites are used in an inconsistent way in literature. Therefore, we introduce the freely accessible MS website for low molecular mass spider venom metabolites, venoMS, containing structural information, MS/MS spectra, and links to related literature. Currently the database contains the structures of 409 acylpolyamine toxins, 36 free linear polyamines, and 81 additional spider venom metabolites. Implemented into this website is a fragment ion calculator (FRIOC) that allows us to predict fragment ions of linear polyamine derivatives. With three metabolites from the venom of the spider Agelenopsis aperta, it was demonstrated how the new website can support the structural elucidation of acylpolyamines using their MS/MS spectra.