title: High-density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single-chain IgE antibody fragments obtained by combinatorial cloning.
abstract: The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ∼25-30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen-allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens' C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients' polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.
authors: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; and.
authors: Christoph
authors: C
authors: Madritsch
authors: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; and.
authors: Elisabeth
authors: E
authors: Gadermaier
authors: GE Healthcare Europe GmbH, 79111 Freiburg, Germany.
authors: Uwe W
authors: UW
authors: Roder
authors: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; and.
authors: Christian
authors: C
authors: Lupinek
authors: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; and.
authors: Rudolf
authors: R
authors: Valenta
authors: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria; and sabine.flicker@meduniwien.ac.at.
authors: Sabine
authors: S
authors: Flicker
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010449:Peptide Mapping; D031784:Phleum / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D020349:Surface Plasmon Resonance
chemical_list: D000485:Allergens; D019151:Peptide Library; D010940:Plant Proteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C090396:PHLPI protein, Phleum pratense; D007073:Immunoglobulin E
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