Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients

PLoS One. 2014 Oct 29;9(10):e111041. doi: 10.1371/journal.pone.0111041. eCollection 2014.


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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allergens / immunology*
  • Allergens / therapeutic use
  • Animals
  • Antibodies / immunology
  • Dander / chemistry
  • Dander / immunology
  • Female
  • Humans
  • Hypersensitivity / diagnosis
  • Hypersensitivity / immunology*
  • Hypersensitivity / therapy
  • Immunoglobulin E / immunology
  • Immunotherapy
  • Lipocalins / immunology*
  • Lipocalins / therapeutic use
  • Mice
  • Mice, Inbred BALB C
  • Protein Multimerization*


  • Allergens
  • Antibodies
  • Can f 4 allergen, Canis familiaris
  • Lipocalins
  • Immunoglobulin E

Grants and funding

This research was funded by grants from the Erling-Persson Family Foundation, the Swedish Asthma and Allergy Association's Research Foundation, the Cancer and Allergy Foundation, the Konsul Th C Bergh Foundation, the Hesselman Foundation, the Swedish Research Council, the Swedish Heart-Lung Foundation, the Stockholm County Council, the Centre for Allergy Research, the European Commission (the 7th Framework Program) BioStruct-X project (contract number 283570) and Karolinska Institutet. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.