Characterization of Phl p 1-specific IgE

Recombinant allergens were purchased from Biomay (Vienna, Austria). The Phl p 1–specific IgE Fab clone 25 was isolated from a combinatorial library in a previous study (19 (link)) (Table I). The V regions of this clone were also grafted onto a complete human monoclonal IgG₁ Ab designated P1 IgG₁ and used to engineer ScFv 25 H chain variable (V_H)/25 L chain variable (V_L) (14 (link), 20 (link)). Serum samples from grass pollen–allergic patients were analyzed with approval of the Ethics Committee of the Medical University of Vienna in a retrospective and anonymized manner. The diagnosis of grass pollen allergy was based on a patient’s history of seasonal symptoms, skin prick testing, and allergen-specific IgE measurements as described (5 (link)). Grass pollen–allergic patients’ sera were also tested for IgE reactivity toward the four major recombinant grass pollen allergens, that is, Phl p 1, Phl p 2, Phl p 5, and Phl p 6, by ELISA. A comprehensive IgE reactivity profile toward >100 purified allergen molecules was established using ImmunoCAP ISAC assays (Thermo Fisher Scientific, Uppsala, Sweden) (21 (link)) (Supplemental Table I). Phl p 1–specific IgE Ab levels in patients’ sera were determined by ImmunoCAP g205 (Thermo Fisher Scientific). Phl p 1–derived peptides were synthesized and purified as described (22 (link)).