2.1 Study participants
Allergic subjects were recruited from the Allergy Clinics of The Alfred and Box Hill Hospitals Melbourne, Victoria, Australia (Alfred Ethics Committee projects 509/11 and 514/13). Forty-one BV-allergic patients (18-68 years; 32% female) were diagnosed on the basis of a systemic allergic response to a bee sting and serum BV-specific IgE of ≥0.35 kUA/L (ImmunoCAP, Phadia, Uppsala, Sweden). Fifty RGP-allergic patients (18-65 years; 52% female) were included with moderate to severe seasonal allergic rhinitis with or without asthma, and serum RGP-specific IgE of ≥0.35 kUA/L (ImmunoCAP). Blood from RGP-allergic subjects was collected in April/May outside of the Australian grass pollen season (Sept-Dec). Twenty-six control subjects (22-58 years; 50% female) had no clinical history of BV and/or RGP allergy as appropriate for the assay, and no detectable specific IgE to the relevant allergen (Monash University project 2016-0289). Exclusion criteria were immunodeficiency, AIT within the last five years, and treatment with continuous oral corticosteroids and/or β-blockers. The use of symptomatic medications (incl. antihistamines and topical corticosteroids) for allergic rhinitis was permitted. The study was conducted according to the principles of the Declaration of Helsinki, and written informed consent from each participant was obtained prior to inclusion.
2.2 Blood sampling and ELISA
Heparinized blood samples were processed within 24 hours of collection for basophil activation, PBMC and serum isolation and storage. Serum RGP-specific-IgE and BV-specific IgE levels were measured by ImmunoCAP using allergen extracts as per manufacturer’s instructions at the Alfred Pathology Services (Alfred Hospital, Melbourne, Australia). Serum Lol p 1- and Lol p 5-specific IgE were measured by a semi-quantitative in-house ELISA as described previously.24,30,31Briefly, ELISA plate wells were coated with recombinant monomeric, non-biotinylated Lol p 1 (MyBiosource, San Diego, CA, US) or Lol p 5 (see below), blocked with 5% skim milk powder in PBS and incubated with serial dilutions of serum samples. Separate wells were incubated with a range of concentrations of purified recombinant human IgE (clone AbD18705; Bio-Rad, Puchheim, Germany) to generate a standard curve for quantification of IgE in serum. Bound IgE was detected using polyclonal rabbit anti-hIgE (Agilent, Santa Clara, CA, US) followed by polyclonal goat anti-rabbit HRP (Promega, Madison, WI, US). ELISA were developed using TMB (Thermo Fisher Scientific, Waltham, MA, US) before the reaction was stopped with 1M HCl and absorbance measured at OD 450 nm on a Multiskan Microplate Spectrophotometer (Thermo Fisher Scientific). Wells without allergens were used to determine background values that were subtracted from allergen-specific IgE values and results are expressed in arbitrary units (AU).