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 â¼25-30 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.
Subject(s)
Allergens/immunology , Immunoglobulin E/immunology , Plant Proteins/immunology , Pollen/immunology , Single-Chain Antibodies/immunology , Allergens/genetics , Amino Acid Sequence , Antibody Affinity , Binding Sites, Antibody , Cells, Cultured , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Immunoglobulin E/genetics , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Models, Molecular , Molecular Sequence Data , Peptide Library , Peptide Mapping , Phleum/chemistry , Phleum/immunology , Plant Proteins/genetics , Pollen/chemistry , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Single-Chain Antibodies/genetics , Surface Plasmon ResonanceABSTRACT
Ubiquitin family modifiers (UbFs) are protein-protein interaction modules acting within a variety of cellular processes. In combination with other techniques, surface plasmon resonance (SPR)-based technology has been used to characterize the interactions of UbFs with their binding partners. SPR binding assays allow the real-time detection of binding events with unlabeled analytes, yet are often hindered by the requirement for careful sample preparation and optimized assay conditions. This chapter aims to share our experience in SPR analysis of UbFs and provide helpful hints in sample preparation, experimental design, evaluation, and data interpretation.
