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

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

BMC biotechnology (2015-12-03)
Valerie Stadlmann, Hanna Harant, Irina Korschineck, Marcela Hermann, Florian Forster, Albert Missbichler
ABSTRACT

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. 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. ScFv can be of benefit for future CD treatment regimes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Isopropyl β-D-thiogalactopyranoside solution, ReadyMade IPTG solution for Blue-white screening
Sigma-Aldrich
Ethanol, ACS reagent, prima fine spirit, without additive, F15 o1
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, HPLC/spectrophotometric grade
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, meets USP testing specifications
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, for molecular biology
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, for molecular biology
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
Ethanol, puriss. p.a., absolute, ≥99.8% (GC)
SAFC
Isopropyl β-D-1-thiogalactopyranoside
Sigma-Aldrich
Kanamycin sulfate from Streptomyces kanamyceticus, Animal Component-free
Sigma-Aldrich
Isopropyl β-D-1-thiogalactopyranoside, ≥99% (TLC)
Sigma-Aldrich
Kanamycin sulfate from Streptomyces kanamyceticus, meets USP testing specifications, powder
Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
L-Lysine monohydrochloride, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Kanamycin sulfate from Streptomyces kanamyceticus, powder, BioReagent, suitable for cell culture, suitable for plant cell culture
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
L-Lysine monohydrochloride, BioUltra, ≥99.5% (AT)
Sigma-Aldrich
Imidazole buffer Solution, BioUltra, 1 M in H2O
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, meets USP testing specifications
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
Imidazole, ReagentPlus®, 99%, Redi-Dri, free-flowing
Sigma-Aldrich
Imidazole, for molecular biology, ≥99% (titration), free-flowing, Redi-Dri
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥98% (TLC)
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, tablet, 1 mg substrate per tablet
Sigma-Aldrich
Ethanol Fixative 80% v/v, suitable for fixing solution (blood films)
Supelco
Ethanol standards 10% (v/v), 10 % (v/v) in H2O, analytical standard
Sigma-Aldrich
Octanoic acid, ≥99%
Sigma-Aldrich
Sodium chloride solution, 5 M