A 45-yr-old male had complained of recurrent wheezing and dyspnea for 7 months. He had worked in a herbal manufacturing factory for 8 yr where he classified many kinds of herbal materials, including Wonji, to prepare for processing. He had experienced nasal symptoms including profuse rhinorrhea and sneezing 4 yr after starting work. These symptoms were aggravated when handling Wonji in the manufacturing process, but improved during holidays and vacations. He was a non-smoker, and had no relative with asthmatic symptoms. His serum total IgE level was 663 U/mL using fluorescent enzyme immunoassay, and the total eosinophil count in the blood was 578/microL. A skin prick test with 55 common aeroallergens showed immediate positive responses to Dermatophagoides pteronyssinus, Dermatophagoides farinae, tree pollens, and grass pollens. Methacholine bronchial challenge test revealed 20% decline of FEV1 at the methacholine concentration of 1.9 mg/mL.
Wonji was obtained from the patient's employer and was prepared as previously described. In brief, Wonji was cut into small pieces and extracted into phosphate-buffered saline (PBS; pH 7.5) 1:5 wt/vol, at 4C for 24 hr, followed by centrifugation at 15,000 rpm at 4C for 30 min. The supernatant was dialyzed (the cut-off molecular weight was 6 kDa) against 4L of normal saline at 4C for 48 hr and then used as crude extracts. The protein concentration of the extracts was determined by bicinchoninic acid (BCA) assay according to the manufacturer's instructions (Pierce, Rockford, IL, U.S.A.). The extract was used for the skin prick tests and specific bronchial challenge test.
One mL of normal saline was administered from a DeVilbiss 646 nebulizer (CS & M Instrument Co., Doylestown, PA, U.S.A.) connected to a dosimeter. The subject was asked to breathe the aerosol with tidal breathing, and this was followed by Wonji extract. The forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were measured using a spirometer (Multispiro-SX , Irvine, CA, U.S.A.) before and 10 min after each inhalation, every 10 min during the first hour, and then every hours for the next 8 hr after the challenge.
The presence of specific IgE antibody to Wonji extract was determined by ELISA, as preciously described. In brief, microtiter plates (NUNC; immunoplate, Roskilde, Denmark) were coated with 50 microL of Wonji extract (100 microg/mL), and then incubated with 50 microL of either the patient's serum, or undiluted sera from five asthmatics who showed negative skin prick test responses to common aeroallergens as well as Wonji extract. After washing, the immunoplate was incubated with 1:1,000 vol/vol biotin-labeled goat anti-human IgE antibody (Sigma, St. Louis, MO, U.S.A.) followed by incubation with 1:1,000 vol/vol streptavidin-peroxidase (Sigma). After washing, 75 microL of TMB solution, 3,3',5,5'-tetramethylbenzidine, one tablet in 10 mL of phosphate citrate buffer containing 2 microL of 30% hydrogen peroxide was added as substrate, and 75 microL of 2.5 N H2SO4 was added to stop the reaction 5 min later. A calorimetric reaction was measured by the absorbency at 450 nm on an ELISA reader. All assays were performed in triplicate.
Competitive ELISA inhibition tests were performed to determine the specificity of IgE binding to a Wonji antigen. In brief, 50 microL of the patient's serum were preincubated with Wonji extract and Der p 2, mugwort extracts for 1 hr at room temperature. The mixture was then incubated on a Wonji-coated microtiter plate for 2 hr. The same steps were then followed as for ELISA. After studying the control samples, in which equal volumes of PBS were preincubated instead of inhibitors, the inhibition of the specific IgE binding was expressed as: 100-(absorbance of samples preincubated with allergens/absorbance of samples preincubated with PBS)x100 (%).
The protein composition pattern and specific IgE binding components of Wonji were analyzed by SDS-PAGE and immunoblot analysis by using the patient's serum. In brief, 25 microg of Wonji extracts were loaded and separated by 12% SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 solution (Bio-Rad, Hercules, CA, U.S.A.) and analyzed. To identify the specific IgE binding components of Wonji extracts, immunoblot analysis was performed with the asthmatic patient's serum. After separating the proteins by SDS-PAGE, they were electrophoretically transferred from the gel to a nitrocellulose membrane in a Bio-Rad Trans-Blot system. Blocking was done by incubation in a solution of 10% non-fat dried milk in 0.05% TBS-T buffer, pH 7.5 for 1 hr at room temperature. The nitrocellulose membrane was then washed, cut into strips and separately incubated overnight at 4C, with the patient's serum which had been diluted 1:10 with the blocking solution. The membrane was then washed and incubated with goat anti-human IgE conjugated HRP (Sigma, St. Louis, MO, U.S.A.), in the presence of blocking solution, for 1 hr at room temperature. After further washing, the membrane was incubated in SuperSignalWest Pico chemiluminescent substrate (Pierce, Rockford, IL, U.S.A.) for 5 min. Fluorescence signals were detected by autoradiography using the Kodak Biomax Light ML film (Eastman Kodak Company, Rochester, NY, U.S.A.).