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Human IgE against the major allergen Bet v 1--defining an epitope with limited cross-reactivity between different PR-10 family proteins.

Levin M, Davies AM, Liljekvist M, Carlsson F, Gould HJ, Sutton BJ, Ohlin M - Clin. Exp. Allergy (2014)

Bottom Line: The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation.We here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response.Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, Lund University, Lund, Sweden.

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Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.
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fig04: Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.

Mentions: To map the epitopes of the available scFv-CHε2-4 fusion proteins, their ability to bind to a set of peptides covering the amino acid sequence of Bet v 1.0101 was assessed by ELISA (Figure S4). Clone M0418 showed strong and B14 showed weak reactivity to a peptide covering residues I56 to D69, while B13 bound weakly to a peptide covering residues G26 to S39. B10 failed to bind any of the tested peptides, but, as mentioned above, should likely bind the same epitope as B13. These binding specificities were confirmed by testing the ability of the identified peptides in soluble form to inhibit binding to immobilized Bet v 1 (Fig.4a).


Human IgE against the major allergen Bet v 1--defining an epitope with limited cross-reactivity between different PR-10 family proteins.

Levin M, Davies AM, Liljekvist M, Carlsson F, Gould HJ, Sutton BJ, Ohlin M - Clin. Exp. Allergy (2014)

Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215112&req=5

fig04: Peptide mapping of epitopes recognized by Bet v 1-specific scFv-Fcε fusion proteins and reactivity profiling of IgE clone M0418. A peptide encompassing residues I56 to D69 of Bet v 1.0101, recognized by the two sequence-related IgE clones M0418 and B14, and an additional peptide spanning residues G26 to S39, recognized by B10, had been identified (Figure S4). Inhibition of binding of these scFv-Fcε to immobilized Bet v 1 by respective peptides confirmed the reactivity of these scFv-Fcε to these parts of the allergen (a). Alanine-scanning modifications of residues within this sequence defines the importance of each residue for reactivity with M0418 (b). Further, reactivity profile of M0418 against the identified Bet v 1.0101 peptide I56 to D69 and modified versions thereof was performed (c). Modifications include some of those found in isoforms of Bet v 1, in homologues of Bet v 1 found in other Betula species, or in PR-10 family proteins of other plant species. Critical amino acid differences that explain an inability of M0418 to bind different isoforms/homologues are apparent, as illustrated by the lack of reactivity (d) against Mal d 1.0108 and Aln g 1.0101 (both of which among other differences carry the F62S mutation). Samples were run in duplicate and error bars represent one standard deviation.
Mentions: To map the epitopes of the available scFv-CHε2-4 fusion proteins, their ability to bind to a set of peptides covering the amino acid sequence of Bet v 1.0101 was assessed by ELISA (Figure S4). Clone M0418 showed strong and B14 showed weak reactivity to a peptide covering residues I56 to D69, while B13 bound weakly to a peptide covering residues G26 to S39. B10 failed to bind any of the tested peptides, but, as mentioned above, should likely bind the same epitope as B13. These binding specificities were confirmed by testing the ability of the identified peptides in soluble form to inhibit binding to immobilized Bet v 1 (Fig.4a).

Bottom Line: The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation.We here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response.Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, Lund University, Lund, Sweden.

Show MeSH