Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5

Cite this as: C. Madritsch, S. Flicker,S. Scheiblhofer, D. Zafred, T. Pavkov-Keller, J. Thalhamer, W. Keller and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 270–280. 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 IgG1 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 IgG1 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. Conclusion and Clinical Relevance 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.