title: Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.
abstract: BACKGROUND
  Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.
  
  
  OBJECTIVE
  To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.
  
  
  METHODS
  A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.
  
  
  RESULTS
  The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.
  
  
  CONCLUSIONS
  We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.
authors.affiliation: Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden; Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
authors.forename: Ola B
authors.initials: OB
authors.lastname: Nilsson
authors.affiliation: Department of Clinical Neuroscience, Therapeutic Immune Design Unit, Center for molecular Medicine (CMM), Karolinska Institutet, Stockholm, Sweden.
authors.forename: Theresa
authors.initials: T
authors.lastname: Neimert-Andersson
authors.affiliation: Department of Clinical Neuroscience, Therapeutic Immune Design Unit, Center for molecular Medicine (CMM), Karolinska Institutet, Stockholm, Sweden.
authors.forename: Mattias
authors.initials: M
authors.lastname: Bronge
authors.affiliation: Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Grundström
authors.affiliation: Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Ranjana
authors.initials: R
authors.lastname: Sarma
authors.affiliation: Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Hannes
authors.initials: H
authors.lastname: Uchtenhagen
authors.affiliation: European Molecular Biology Laboratory (EMBL) - Hamburg Outstation, c/o DESY, Hamburg, Germany.
authors.forename: Alexey
authors.initials: A
authors.lastname: Kikhney
authors.affiliation: Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Sandalova
authors.affiliation: Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden; Department of Clinical Neuroscience, Therapeutic Immune Design Unit, Center for molecular Medicine (CMM), Karolinska Institutet, Stockholm, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Holmgren
authors.affiliation: European Molecular Biology Laboratory (EMBL) - Hamburg Outstation, c/o DESY, Hamburg, Germany.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Svergun
authors.affiliation: Science for Life Laboratory, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Adnane
authors.initials: A
authors.lastname: Achour
authors.affiliation: Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Marianne
authors.initials: M
authors.lastname: van Hage
authors.affiliation: Department of Clinical Neuroscience, Therapeutic Immune Design Unit, Center for molecular Medicine (CMM), Karolinska Institutet, Stockholm, Sweden.
authors.forename: Hans
authors.initials: H
authors.lastname: Grönlund
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000627:therapeutic use; D000818:Animals; D000906:Antibodies / Q000276:immunology; D063607:Dander / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis / Q000276:immunology* / Q000628:therapy; D007073:Immunoglobulin E / Q000276:immunology; D007167:Immunotherapy; D054834:Lipocalins / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D055503:Protein Multimerization
chemical_list: D000485:Allergens; D000906:Antibodies; C000590661:Can f 4 allergen, Canis familiaris; D054834:Lipocalins; D007073:Immunoglobulin E