title: Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the Timothy grass major allergen Phl p 5a.
abstract: The scarcity of monoclonal human IgE antibodies with specificity for defined allergens is a bottleneck for the molecular characterisation of allergens and their epitopes. Insights into the characteristics of such antibodies may allow for analyses of the molecular basis underlying allergenicity and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain and the IgG1, IgG4, IgE, IgA1, and IgA2 heavy chains, the corresponding human immunoglobulins were constructed and produced in mammalian cells. In parallel, a murine hybridoma line with specificity for Phl p 5a was established, recloned and produced as human chimeric IgE. After purification, immunoreactivity of the antibodies with the allergen was assessed. Applicability in allergy diagnostics was confirmed by establishment of artificial human sera. Functionality of both antibodies was further demonstrated in receptor binding studies and mediator release assays using humanised rat basophil leukaemia cells (RBL-SX38) suggesting the presence of spatially separate epitopes. By using Phl p 5 fusion proteins and recombinant IgE in immunoblotting and mediator release assays we assigned the epitope of the authentic IgE to a looped stretch exclusively present in Phl p 5a. In summary, the Phl p 5-specific antibodies are the first full set of allergy-related antibody isotypes of their kind and represent valuable tools for studies of fundamental mechanisms and structure/function relationships in allergy.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Department of Chemistry, University of Hamburg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Hecker
authors.forename: Andrea
authors.initials: A
authors.lastname: Diethers
authors.forename: Stefanie
authors.initials: S
authors.lastname: Etzold
authors.forename: Henning
authors.initials: H
authors.lastname: Seismann
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Michel
authors.forename: Melanie
authors.initials: M
authors.lastname: Plum
authors.forename: Reinhard
authors.initials: R
authors.lastname: Bredehorst
authors.forename: Simon
authors.initials: S
authors.lastname: Blank
authors.forename: Ingke
authors.initials: I
authors.lastname: Braren
authors.forename: Edzard
authors.initials: E
authors.lastname: Spillner
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000276:immunology*; D051379:Mice; D008863:Microspheres; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology; D031784:Phleum / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology*; D051381:Rats; D011961:Receptors, Fc / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007132:Immunoglobulin Isotypes; D050505:Mutant Proteins; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011961:Receptors, Fc; D011994:Recombinant Proteins; D007073:Immunoglobulin E