Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease
| Autor: | Florian Forster, Marcela Hermann, Irina Korschineck, Albert Missbichler, Hanna Harant, Valerie Stadlmann |
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| Rok vydání: | 2015 |
| Předmět: |
Genetic Vectors
Enzyme-Linked Immunosorbent Assay chemical and pharmacologic phenomena Disease Single chain digestive system Gliadin scFv law.invention law Blocking antibody Animals Lymphocytes Dietary gluten chemistry.chemical_classification biology nutritional and metabolic diseases food and beverages respiratory system Gluten digestive system diseases Celiac Disease Celiac disease treatment chemistry Wheat Immunology biology.protein Recombinant DNA Chickens Single-Chain Antibodies Research Article Prolamins Protein Binding Biotechnology |
| Zdroj: | BMC Biotechnology |
| ISSN: | 1472-6750 |
| DOI: | 10.1186/s12896-015-0223-z |
| Popis: | Background Celiac 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. Results Gluten-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. Conclusion ScFv can be of benefit for future CD treatment regimes. |
| Databáze: | OpenAIRE |
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