Reagents and plasma
A cat major allergen, Fel d 1, and a fungal major allergen, Asp f 1, were purchased
from INDOOR Biotechnologies (Charlottesville, VA, USA). Crude fungal extract (CFE)
was purchased from ITEA (Tokyo, Japan). All chemicals were purchased from Katayama
Chemical Industries (Osaka, Japan), Sigma-Aldrich (St. Louis, MO, USA) or Nacalai
Tesque (Kyoto, Japan) unless otherwise indicated. Plasma was obtained from two cat-allergic
asthma patients who exhibited positive CAP-RAST score to Fel d 1 and from three fungi-allergic
asthma patients who showed positive CAP-RAST score to Asp f 1. The plasma were stored
at ?30 °C until use. All studies that used human samples were approved by the institutional
ethics committee at Showa University School of Medicine (Tokyo, Japan).
Treatment of positively- and negatively-charged plasma cluster ions
Airborne allergens; Fel d 1, CFE and Asp f 1, were treated with positively- and negatively-charged
plasma cluster ions (PC-ions), as described previously 3]. Briefly, a cylindrical apparatus (14.5 cm diameter?×?52.5 cm height) was filled
with PC-ions (2.5?×?10
4
PC-ions/cm
3
) for 6 h, and then 221 mL of 1 mg/ml Fel d 1 solution or 666 ml of 40 ng/ml CFE or
Asp f 1 solution was nebulized from top of the apparatus to expose the PC-ions. Nebulized
solution was collected in a 10 cm petri dish (BD Biosciences, San Diego, CA, USA)
that was placed underneath the apparatus (Fig. 1). Sham-treated allergen was prepared in the same condition, but without generating
the PC-ions.
Fig. 1. Cylindrical apparatus for PC-ions exposure. PC-ions were generated in four Plasma
cluster™ devices (shown as gray boxes) that were placed inside of wall of the apparatus. PC-ions were emitted from the
devices and filled the apparatus at 2.5?×?10
4
ions/cm
3
. A “mist” solution containing allergen was generated from a nebulizer located on
the top of the apparatus, which is shown in a box with horizontal stripe. The solution
was shed at 2.5 L/min (flow rate) and 3.3 m/s (linear velocity). A petri dish was
placed beneath the apparatus to collect the PC-ions- or sham-treated allergen solution
Quantification of Fel d 1 and Asp f 1 by ELISA and mass spectrometric analysis
Amount of Fel d 1 and Asp f 1 was quantified using a Fel d 1- or Asp f 1-specific
enzyme-linked immunosorbent assay (ELISA) kit (INDOOR Biotechnologies) following the
manufacturer’s instructions. Prior to quantification of Fel d 1 by ELISA, CFE and
Fel d 1 solution after PC-ions or sham treatment were 1,250-fold concentrated by lyophilization.
Fel d 1 that was collected after PC-ions or sham treatment was separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition
and proteins were visualized by silver staining following the manufacturer’s instructions
(Cosmo Bio, Tokyo, Japan). Proteins separated on SDS-PAGE were identified by liquid
chromatography (LC) connected to a mass spectrometer (MS, LTQ Orbitrap XL, Thermo
Fisher Scientific, Rockford, IL, USA) using a HPLC column (Develosil ODS-HG-5, Nomura
Chemical, Aichi, Japan) and Xcalibur software v2.0.7 (Thermo Fisher Scientific), as
described elsewhere 16].
ELISA inhibition
The immunoglobulin E (IgE)-binding capacity of PC-ions-treated allergens for pooled
plasma from patients sensitized with each allergen was analyzed using an ELISA inhibition
assay, as described previously with slight modifications 3]. Briefly, a 96-well microtiter plate (NUNC maxisorp, Thermo Fisher scientific) was
coated with 0.1 ml of 200 ng/ml sham-treated Fel d 1, CFE or Asp f 1 dissolved in
100 mM bicarbonate buffer (pH 9.4) overnight at 4 °C. After washing three times with
phosphate buffered saline containing 0.05 % Tween 20 (PBST), plate were blocked with
PBST containing 3 % skim milk and 1 % bovine serum albumin (BSA) for 2 h. Pooled plasma
was diluted 80-fold from two Fel d 1-positive asthmatic patients or 12.5-fold from
three Asp f 1-positive asthmatic patients, which was preincubated with serial dilutions
of PC-ions- or sham-treated Fel d 1, CFE or Asp f 1 overnight at 4 °C. Then, pooled
plasma was applied to sham-treated Fel d 1-, CFE- or Asp f 1-coated plates, which
were incubated for 12 h at 4 °C. After washing with PBST three times, plates were
incubated with biotinylated anti-human IgE monoclonal antibody (Vector Laboratories,
Burlingame, CA, USA) diluted 1:1,000 for 1 h at room temperature, followed by 1 h
incubation with alkaline phosphate-conjugated streptavidin (Jackson ImmunoResearch
Laboratories, West Grove, PA, USA). To determine the amount of IgE-binding, Attophos
substrate solution (Promega, Fitchburg, WI, USA) was added to the plate and fluorescence
intensity was measured using a Wallac 1420 ARVOsx Multilabel Counter (Perkin-Elmer,
Downers Grove, IL, USA).
Statistical analysis
Statistical analyses were performed using non-repeated ANOVA. For differences that
were found to be significant, post-hoc analysis using a Student-Newman-Keuls test
was performed. A threshold for statistical significance was set at P??0.01.