Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease

Abstract

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.

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

“…E. coli BL21(DE3) (Life technologies, Carlsbad, CA, USA) and the pET28a(+) expression system were used for production of the recombinant scFv which describes an engineered IgY fragment against PT-gliadin useful for the treatment of celiac disease (Stadlmann et al 2015 ).…”

“…In this study, we used E. coli BL21(DE3) and the pET expression system for the production of a novel scFv (Stadlmann et al 2015 ). We hypothesized that induction by lactose increases the amount of soluble product compared to IPTG.…”

A combination of HPLC and automated data analysis for monitoring the efficiency of high-pressure homogenization

“…All experiments were performed with a recombinant E.coli BL21(DE3) strain producing a recombinant single chain fragment variable (scFv) against gliadin, which causes coeliac disease [ 26 ].…”

The production of a recombinant tandem single chain fragment variable capable of binding prolamins triggering celiac disease

“…Even in “gluten-free” dietary products traces of prolamins are found, which have a severe impact on the well-being [ 10 ]. To support those patients we recently developed a novel single chain Fragment variable (scFv) against prolamins [ 11 ]. This scFv works as a “neutralizing agent”, meaning that a complex between prolamin and the scFv is formed in the gut and no systemic interactions are expected, as the formed complex does not cross the epithelial barrier and is finally excreted.…”

“…To obtain this novel scFv, we immunized chicken with peptic tryptic digested gliadin (PT-gliadin). Those immunized chicken were used as source for RNA, carrying the sequence for the recombinant scFv [ 11 ]. Since no effector function of the antibody (AB) is relevant for the neutralizing effect, but only the variable light and heavy chain are required, we generated a single chain Fragment variable (scFv).…”