Two individuals with histories of allergy to D. batatas were enrolled. Regarding clinical history, one patient had severe urticaria and angioedema from eating fresh D. batatas powder, and the other patient, who had been working in a herbal shop as a herbal merchant, complained of cough, wheezing, and dyspnea upon exposure to D. batatas dust. Skin prick testing with common inhalant allergens, food allergens, and D. batatas extracts was performed. The results of the skin prick tests are expressed as the ratios of mean wheal diameter of allergen to histamine (A/:H ratio).

Patient 1 was a 26-yr-old female, who complained of severe urticaria and angioedema following indigestion of D. batatas with water as a health food. She had been suffering from multiple food allergies including shellfish and peaches, as well as allergic rhinitis.

Patient 2 was a 29-yr-old female who had been working as a merchant dealing in several herbal materials. She presented at the emergency department with sudden onset of dyspnea following exposure to D. batatas dust. Patient 2 had allergies to foodstuffs, including chestnuts and potatoes, as well as allergic rhinitis.

D. batatas powder was purchased at a local market and was extracted with phosphate-buffered saline (PBS [pH7.5], 1:5 w/v) at 4℃ overnight. Then it was centrifuged at 10,000 RPM at 4℃ for 30 min, and supernatant was dialyzed against 2L of PBS at 4℃ for 48 hr and then used for the enzyme-linked immunosorbent assays (ELISAs), immunoblot analysis, and 2-dimensional electrophoresis. For the skin prick tests, the supernatants were mixed with an equal amount of sterile glycerin.

Airway responsiveness to methacholine was tested using the 5-breath dosimeter protocol described previously (5). Bronchoprovocation tests were performed according to the procedure used in previous occupational asthma studies (3). The concentrations of inhaled antigen extracts ranged from 1:1,000 w/v to 1:10 w/v.

The presence of specific antibodies to D. batatas extracts was determined by ELISA using a modified method as described previously (2). A 96-well ELISA plate (Corning, Action, MA, U.S.A.) was coated with 1 µg of antigen. The sera of two patients and eighteen non-atopic healthy controls were 1:2 diluted for specific IgE antibody, and 1:10 diluted for specific IgG1 and IgG4 antibodies. The presence of serum specific IgE, IgG1, and IgG4 antibodies was determined by positive cut-off values, which were derived from the mean plus three standard deviations of readings for the sera of the healthy controls.

D. batatas extract (0.6 µg/well) were applied to a Cambrex precast Tris-glycine homogenous gel (4-20% acrylamide). Electrophoresis was performed with a Novex Mini-cell (Novex, San Diego, CA, U.S.A.) for 90 min at 130 constant voltages. The gel was fixed and stained with Coommassie Brilliant Blue. For immunoblotting the proteins of the gel was transferred to polyvinylidine difluoride membrane (Millipore, Billerica, MA, U.S.A.), which was then treated with a 0.5% fetal bovine serum-Tris-buffered saline solution for 1 hr to block nonspecific protein binding. The membrane was then incubated with the 1:1 vol:vol diluted sera (with TBS) for 2 hr at room temperature, and then washed with TBS with 0.1% Tween-20 (TBS-Tween). Bound specific IgE was detected by biotin-conjugated anti-human IgE antibody (1:1,000 vol/vol, Vector Laboratories Inc.) conjugated with streptavidin alkaline phosphatase (1:1,000 vol/vol, Sigma-Aldrich) followed by the substrate solution (NBT/BCIP kit, Sigma-Aldrich).

2D gel electrophoresis was performed using a modified method as described previously with D. batatas extracts (15 µg per well) (6).

To confirm the major allergenic components via N-terminal sequencing, the 2D gel electrophoresed proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane. The protein spots were excised and micro-sequencing was performed using the Procise 492 c1c protein sequencer (Applied Biosystems, Foster City, CA, U.S.A.).

Clinical features of two subjects are demonstrated in Table 1.

In patient 1, skin prick test showed positive responses to Dermatophagoides pteronyssinus (6.0), Dermatophagoides farinae (3.25), cockroach (2.0), and D. batatas (4.5). Airway hyperresponsiveness to methacholine was confirmed at PC₂₀, 3.563 mg/mL. Oral provocation test with D. batatas could not be done because she had certain histories of severe food allergy reactions twice and refused the provocation test. In patient 2, skin prick test showed positive responses to D. pteronyssinus (1.65), alder (4.4), birch (0.85), and D. batatas (1.0). Airway hyperresponsiveness to methacholine was confirmed at PC₂₀, 0.75 mg/mL, and the bronchoprovocation test to D. batatas extracts (1:1,000 w/v) showed early asthmatic response.

The levels of serum-specific IgE antibodies to D. batatas extracts were significantly higher in the two patients than in non-atopic healthy controls. Serum-specific IgG1 and IgG4 antibodies were also detected in the sera from the two patients (Fig. 1).

Fig. 2 shows the result of the SDS-PAGE and IgE immunoblot analyses of the D. batatas extracts. A 27-kDa IgE-binding component was detected in the sera of the two patients but not in the serum of the non-atopic healthy control. Fig. 3 shows the results of the 2D electrophoresis and IgE immunoblot. Several spots were noted for the 27-kDa IgE-binding component, and the two largest spots were selected for amino acid sequencing.

Fig. 4 shows the result of the amino acid sequencing analysis. The N-terminal amino acid sequence of the two largest spots in 27-kDa (pI 6.40 and 6.04, respectively) showed 80% of homology with a 268-amino acid tuber storage protein of D. polystachya.