title: Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.
abstract: Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: mueller3@niehs.nih.gov.
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Mueller
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Ankney
authors.affiliation: Indoor Biotechnologies, Inc., Charlottesville, VA 22903, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Glesner
authors.affiliation: U.S. Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Taruna
authors.initials: T
authors.lastname: Khurana
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Lori L
authors.initials: LL
authors.lastname: Edwards
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Lars C
authors.initials: LC
authors.lastname: Pedersen
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Lalith
authors.initials: L
authors.lastname: Perera
authors.affiliation: U.S. Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Jay E
authors.initials: JE
authors.lastname: Slater
authors.affiliation: Indoor Biotechnologies, Inc., Charlottesville, VA 22903, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Pomés
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: London
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D001666:Binding Sites, Antibody / Q000276:immunology; D003058:Cockroaches / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D018360:Crystallography, X-Ray; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008958:Models, Molecular; D009154:Mutation; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000648:ultrastructure*
chemical_list: D000485:Allergens; D000939:Epitopes; D057127:Single-Chain Antibodies; C105482:allergen Bla g 1; C091463:allergen Per a I; D007073:Immunoglobulin E
keywords: Allergen; Bla g 1; Cockroach; Epitope; Structure; scFv