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

Stadlmann V, Harant H, Korschineck I, Hermann M, Forster F, Missbichler A - BMC Biotechnol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Sciotec Diagnostics Technologies GmbH, Tulln, Austria. valeriestadlmann@hotmail.com.

ABSTRACT

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.

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Related in: MedlinePlus

Purified scFv detects PT-Gliadin and natural wheat flour digest. a. ScFv binds to PT-Gliadin in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. b. Purified scFv binds to natural wheat flour digest in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. c. ScFv binding reaches approximate equilibrium after 3 h incubation time. Two single chain concentrations (500 and 1000 ng/ml respectively) were incubated for indicated time and binding to (c). PT-Gliadin or (d). wheat flour digest was assessed by ELISA as described in Methods. Data shown represent OD (450 nm) values at different incubation times compared to the OD (450 nm) value after 3 h (ODx/OD3h *100). Mean values (+/− SEM) of triplicates are shown
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Fig6: Purified scFv detects PT-Gliadin and natural wheat flour digest. a. ScFv binds to PT-Gliadin in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. b. Purified scFv binds to natural wheat flour digest in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. c. ScFv binding reaches approximate equilibrium after 3 h incubation time. Two single chain concentrations (500 and 1000 ng/ml respectively) were incubated for indicated time and binding to (c). PT-Gliadin or (d). wheat flour digest was assessed by ELISA as described in Methods. Data shown represent OD (450 nm) values at different incubation times compared to the OD (450 nm) value after 3 h (ODx/OD3h *100). Mean values (+/− SEM) of triplicates are shown

Mentions: ScFv candidate was purified to homogeneity by immobilized metal affinity and subsequent size-exclusion chromatography. Purified scFv showed concentration -dependent binding to PT-Gliadin as expected (Fig. 6a). Binding reached approximate equilibrium after 3 h of incubation time with scFv (Fig. 6c). We questioned whether scFv would also detect natural wheat antigens, mimicking the real-life situation. Common whole-wheat flour was subjected to gastric digestion in form of “simulated gastric fluid” (SGF), to resemble the protein portion that enters the upper intestinal duodenum after stomach passage. Gastric digested wheat flour, denoted as “wheat flour digest”, was coated onto 96-ELISA plates analogous to PT-Gliadin. Direct ELISA testing revealed concentration -dependent, specific binding of scFv (Fig. 6b), reaching approximate equilibrium after 3 h incubation time (Fig. 6d). Furthermore, scFv detects PT-Gliadin and wheat flour digest under non-reducing and reducing conditions in western blot, revealing similar binding patterns compared to IgY (Fig. 7).Fig. 6


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

Stadlmann V, Harant H, Korschineck I, Hermann M, Forster F, Missbichler A - BMC Biotechnol. (2015)

Purified scFv detects PT-Gliadin and natural wheat flour digest. a. ScFv binds to PT-Gliadin in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. b. Purified scFv binds to natural wheat flour digest in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. c. ScFv binding reaches approximate equilibrium after 3 h incubation time. Two single chain concentrations (500 and 1000 ng/ml respectively) were incubated for indicated time and binding to (c). PT-Gliadin or (d). wheat flour digest was assessed by ELISA as described in Methods. Data shown represent OD (450 nm) values at different incubation times compared to the OD (450 nm) value after 3 h (ODx/OD3h *100). Mean values (+/− SEM) of triplicates are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666168&req=5

Fig6: Purified scFv detects PT-Gliadin and natural wheat flour digest. a. ScFv binds to PT-Gliadin in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. b. Purified scFv binds to natural wheat flour digest in a concentration -dependent manner. Purified scFv was diluted in indicated concentrations and binding was assessed by ELISA as described in Methods. Data shown are OD (450 nm) values and represent mean values (+/− SEM) of triplicates. c. ScFv binding reaches approximate equilibrium after 3 h incubation time. Two single chain concentrations (500 and 1000 ng/ml respectively) were incubated for indicated time and binding to (c). PT-Gliadin or (d). wheat flour digest was assessed by ELISA as described in Methods. Data shown represent OD (450 nm) values at different incubation times compared to the OD (450 nm) value after 3 h (ODx/OD3h *100). Mean values (+/− SEM) of triplicates are shown
Mentions: ScFv candidate was purified to homogeneity by immobilized metal affinity and subsequent size-exclusion chromatography. Purified scFv showed concentration -dependent binding to PT-Gliadin as expected (Fig. 6a). Binding reached approximate equilibrium after 3 h of incubation time with scFv (Fig. 6c). We questioned whether scFv would also detect natural wheat antigens, mimicking the real-life situation. Common whole-wheat flour was subjected to gastric digestion in form of “simulated gastric fluid” (SGF), to resemble the protein portion that enters the upper intestinal duodenum after stomach passage. Gastric digested wheat flour, denoted as “wheat flour digest”, was coated onto 96-ELISA plates analogous to PT-Gliadin. Direct ELISA testing revealed concentration -dependent, specific binding of scFv (Fig. 6b), reaching approximate equilibrium after 3 h incubation time (Fig. 6d). Furthermore, scFv detects PT-Gliadin and wheat flour digest under non-reducing and reducing conditions in western blot, revealing similar binding patterns compared to IgY (Fig. 7).Fig. 6

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Sciotec Diagnostics Technologies GmbH, Tulln, Austria. valeriestadlmann@hotmail.com.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus