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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

ScFv binds to digests of gluten-rich grains, but not to gluten-free grains or pseudo grains. ScFv was diluted to 10, 100, 1000 and 10,000 ng/ml and binding to various grain digests was assessed by ELISA as described in Methods. Data shown were analyzed by 3-parameter curve-fit nonlinear regression using Graph Pad Prism 6 software and are OD values (450 nm) representing the mean (+/− SEM) of triplicates. ScFv detects PT-Gliadin (a), common bread wheat (b), khorasan wheat (c) and to lower degree rye (d). ScFv does not react with barley (e), corn (f), or rice (g). ScFv does not detect the pseudo grain amaranth (h). Please note the axis break in e, f, g and h, which was introduced to demonstrate that OD450 does not exceed 0.2 and represents background signal
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Fig9: ScFv binds to digests of gluten-rich grains, but not to gluten-free grains or pseudo grains. ScFv was diluted to 10, 100, 1000 and 10,000 ng/ml and binding to various grain digests was assessed by ELISA as described in Methods. Data shown were analyzed by 3-parameter curve-fit nonlinear regression using Graph Pad Prism 6 software and are OD values (450 nm) representing the mean (+/− SEM) of triplicates. ScFv detects PT-Gliadin (a), common bread wheat (b), khorasan wheat (c) and to lower degree rye (d). ScFv does not react with barley (e), corn (f), or rice (g). ScFv does not detect the pseudo grain amaranth (h). Please note the axis break in e, f, g and h, which was introduced to demonstrate that OD450 does not exceed 0.2 and represents background signal

Mentions: As we had been able to demonstrate concentration -dependent, specific binding of scFv to PT-Gliadin and wheat flour digest, we wanted to assess scFv reactivity to other grains. Digests of common bread wheat, khorasan wheat, rye, barley, corn, rice and amaranth were coated in four protein concentrations onto 96-well ELISA plates. Four scFv concentrations in the range of 10–10000 ng/ml were subjected to the four antigen-coating concentrations by ELISA, and sigmoid binding curves were analyzed by Graph Pad Prism software. As kinetic binding experiments revealed that approximate equilibrium was reached after a 3 h incubation period with scFv (6 C and D), the incubation time for binding studies was adjusted to this incubation time. Sigmoid binding curves for PT-Gliadin, wheat digest and khorasan wheat digest were comparable (Fig. 9a, b, c), indicating that wheat prolamins are successfully targeted by scFv. Accordingly, scFv detected rye digest (Fig. 9d) to a lower extent than wheat digest. Notably, scFv did not react with barley (Fig. 9e), gluten-free grains like corn (Fig. 9f) and rice (Fig. 9g), or pseudo-grains like amaranth (Fig. 9h). ELISA data were further supported by western blot results, demonstrating scFv binding to PT-Gliadin, wheat flour digest, khorasan wheat flour digest and to a lesser degree to rye. On the contrary, scFv exerts no binding towards barley, corn, rice or amaranth (Fig. 10). Based on data of sigmoid binding curves, we wanted to calculate rough affinity constants for binding of scFv to PT-Gliadin and flour digests. For the calculation we relied on a method for the assessment of mean affinity constants (Kaffmean) by direct ELISA, first described by Loomans et al. in 1995 [26]: Affinity constants were calculated based on the scFv concentration at OD50 of each sigmoid antigen curve. Four scFv concentrations subjected to four antigen-coating concentrations allow three affinity calculations for an antigen coating ratio of two and one for an antigen coating ratio of eight. Mean affinity constants, shown in Table 1, represent the mean of four calculated affinity calculations per antigen. ScFv targets PT-Gliadin, common and khorasan wheat digest with Kaff mean values in the high nanomolar range; 3.82 (+/−1.93) x 107 M-1, 3.21 (+/− 2.14) x 107 M-1 and 2.30 (+/− 1.49) x 107 M-1; respectively.Fig. 9


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)

ScFv binds to digests of gluten-rich grains, but not to gluten-free grains or pseudo grains. ScFv was diluted to 10, 100, 1000 and 10,000 ng/ml and binding to various grain digests was assessed by ELISA as described in Methods. Data shown were analyzed by 3-parameter curve-fit nonlinear regression using Graph Pad Prism 6 software and are OD values (450 nm) representing the mean (+/− SEM) of triplicates. ScFv detects PT-Gliadin (a), common bread wheat (b), khorasan wheat (c) and to lower degree rye (d). ScFv does not react with barley (e), corn (f), or rice (g). ScFv does not detect the pseudo grain amaranth (h). Please note the axis break in e, f, g and h, which was introduced to demonstrate that OD450 does not exceed 0.2 and represents background signal
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig9: ScFv binds to digests of gluten-rich grains, but not to gluten-free grains or pseudo grains. ScFv was diluted to 10, 100, 1000 and 10,000 ng/ml and binding to various grain digests was assessed by ELISA as described in Methods. Data shown were analyzed by 3-parameter curve-fit nonlinear regression using Graph Pad Prism 6 software and are OD values (450 nm) representing the mean (+/− SEM) of triplicates. ScFv detects PT-Gliadin (a), common bread wheat (b), khorasan wheat (c) and to lower degree rye (d). ScFv does not react with barley (e), corn (f), or rice (g). ScFv does not detect the pseudo grain amaranth (h). Please note the axis break in e, f, g and h, which was introduced to demonstrate that OD450 does not exceed 0.2 and represents background signal
Mentions: As we had been able to demonstrate concentration -dependent, specific binding of scFv to PT-Gliadin and wheat flour digest, we wanted to assess scFv reactivity to other grains. Digests of common bread wheat, khorasan wheat, rye, barley, corn, rice and amaranth were coated in four protein concentrations onto 96-well ELISA plates. Four scFv concentrations in the range of 10–10000 ng/ml were subjected to the four antigen-coating concentrations by ELISA, and sigmoid binding curves were analyzed by Graph Pad Prism software. As kinetic binding experiments revealed that approximate equilibrium was reached after a 3 h incubation period with scFv (6 C and D), the incubation time for binding studies was adjusted to this incubation time. Sigmoid binding curves for PT-Gliadin, wheat digest and khorasan wheat digest were comparable (Fig. 9a, b, c), indicating that wheat prolamins are successfully targeted by scFv. Accordingly, scFv detected rye digest (Fig. 9d) to a lower extent than wheat digest. Notably, scFv did not react with barley (Fig. 9e), gluten-free grains like corn (Fig. 9f) and rice (Fig. 9g), or pseudo-grains like amaranth (Fig. 9h). ELISA data were further supported by western blot results, demonstrating scFv binding to PT-Gliadin, wheat flour digest, khorasan wheat flour digest and to a lesser degree to rye. On the contrary, scFv exerts no binding towards barley, corn, rice or amaranth (Fig. 10). Based on data of sigmoid binding curves, we wanted to calculate rough affinity constants for binding of scFv to PT-Gliadin and flour digests. For the calculation we relied on a method for the assessment of mean affinity constants (Kaffmean) by direct ELISA, first described by Loomans et al. in 1995 [26]: Affinity constants were calculated based on the scFv concentration at OD50 of each sigmoid antigen curve. Four scFv concentrations subjected to four antigen-coating concentrations allow three affinity calculations for an antigen coating ratio of two and one for an antigen coating ratio of eight. Mean affinity constants, shown in Table 1, represent the mean of four calculated affinity calculations per antigen. ScFv targets PT-Gliadin, common and khorasan wheat digest with Kaff mean values in the high nanomolar range; 3.82 (+/−1.93) x 107 M-1, 3.21 (+/− 2.14) x 107 M-1 and 2.30 (+/− 1.49) x 107 M-1; respectively.Fig. 9

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