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).