title: Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5.
abstract: BACKGROUND
  Allergen recognition by IgE antibodies is a key event in allergic inflammation.
  
  
  OBJECTIVE
  To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5.
  
  
  METHODS
  In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5.
  
  
  RESULTS
  We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5.
  
  
  CONCLUSIONS
  Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Madritsch
authors.forename: S
authors.initials: S
authors.lastname: Flicker
authors.forename: S
authors.initials: S
authors.lastname: Scheiblhofer
authors.forename: D
authors.initials: D
authors.lastname: Zafred
authors.forename: T
authors.initials: T
authors.lastname: Pavkov-Keller
authors.forename: J
authors.initials: J
authors.lastname: Thalhamer
authors.forename: W
authors.initials: W
authors.lastname: Keller
authors.forename: R
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000485:Allergens / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D006801:Humans; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology*
chemical_list: D000485:Allergens; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E