Serum samples were obtained from patients attending the Allergy-Asthma Clinic at Severance Hospital, Yonsei University College of Medicine in Seoul, Korea. Allergy diagnoses were based on any history of allergic reactions and skin prick testing. Sera from patients were tested for IgE antibodies specific for HDM (Dermatophagoides farinae) allergens using the Uni-CAP system (Phadia, Uppsala, Sweden) and ELISA analysis. Sera from 194 HDM-sensitized subjects (males/females, 103:91; average age, 25.4 years; age range, 4–67 years) and 20 healthy controls were used to assess the IgE reactivity of recombinant proteins. Serum samples were collected after consent of the patients, and experiments using the collected sera were approved by the Institutional Review Board (no. 4-2009-0180). The study was approved by the ethics committee of Sevrance Hospital (Yonsei University College of Medicine).

Frozen Dermatophagoides farinae, Tyrophagus putrescentiae and Blomia tropicalis mites were obtained from the Arthropods of Medical Importance Resource Bank (AMIB) at the Department of Environmental Medical Biology, Yonsei University College of Medicine (Seoul, Korea). Total RNA was isolated from frozen mite bodies using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. First-strand cDNA was synthesized from 5 µg total RNA using avian myeloblastosis reverse transcriptase (Promega, Madison, WI, USA) and an oligo (dT₁₈) primer. Primers were designed based on the published full-length nucleotide sequence of Der f 5 (GenBank accession no. AY283283), Der f 21 (EF027122), Tyr p 5 (AY800358), Tyr p 21 (AY800360), Blo t 5 (U59102) and Blo t 21 (AY800348), and sequences were flanked by restriction enzyme sites for either BamH I/Xho I or BamH I/Sal I. The primer sequences were as follows: Der f 5 forward, 5′-GGATCCATG AAATTCATCATTGCTATTGCTG-3′ and reverse, 5′-CTCGAGTTAAACTTCAATCTTCTTCACACGTTGCTC-3′; Der f 21 forward, 5′-GGATCCATGAAATTCATTATTTTCTGTGCCA-3′ and reverse, 5′-CTCGAGTTAATCATCCGATTTTACAGCTTTCACCTT-3′; Tyr p 5 forward, 5′-GGATCCATGAAGTTCGCCATTCTCGC-3′ and reverse, 5′-CTCGAGTTAGCGAGTCTTGACAGCCT-3′; Blo t 5 forward, 5′-GGATCCATGAAGTTCGCCATCGTT CTTATTG-3′ and reverse, 5′-CTCGAGTTATTGGGTTTG AATATCCTTCACTTTTTG-3′; Tyr p 21 forward, 5′-GGATCCATGAAGTTCGTCATCGCCCT-3′ and reverse, 5′-GTCGACTTAGACCTTGATGGCGTTCACT-3′; Blo t 21 forward, 5′-GGATCCATGAAATTTATCATCGCATTGGCTG-3′ and reverse, 5′-GTCGACTTATTCGGAATCTTGGACTCGCTTT-3′. The primers were synthesized by GeneTech Corporation (Daejeon, Korea). The conditions for PCR amplification were as follows: Pre-denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min and, following the final cycle, an additional extension at 72°C for 5 min. The PCR-amplified cDNA sequences encoding Der f 5, Der f 21, Tyr p 5, 21, Blo t 5 and 21 were cloned into the pCR4-TOPO vector (Invitrogen Life Technologies), sub-cloned into the pET-28a expression vector (Novagen, San Diego, CA, USA), and transformed into E. coli Rosetta™ 2 (DE3) cells (Novagen). The expression of recombinant proteins was induced by addition of 1 mM isopropyl-1-thio-β-galactopyranoside (BioShop Canada Inc., Burlington, ON, Canada), and protein was purified from the insoluble fraction of cell lysates using Ni sepharose excel resin (GE Healthcare, Uppsala, Sweden) under denaturing conditions (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, 6 M urea, pH 7.4). The concentration of each of the recombinant allergens was determined using a Bradford Assay (Bio-Rad Laboratories, Hercules, CA, USA). The purified recombinant allergens were separated by 15% SDS-PAGE, followed by staining with Coomassie Blue R250 (Amresco, Solon, OH, USA).

Recombinant Der f 2 was obtained from the AMIB and used as a control. Microtiter plates were coated with 100 µl recombinant protein (2 µg/ml in 50 mM sodium carbonate, pH 9.6), and washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST). The plates were blocked with 1% bovine serum albumin (BSA; EMD Millipore, Kankakee, IL, USA) in PBST for 1 h at room temperature and then incubated for 1 h with 100 µl serum per well diluted at 1:9 in PBST containing 1% BSA. IgE antibodies were detected using biotinylated goat anti-human IgE (Vector Laboratories, Burlingame, CA, USA) and streptavidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA). The assay was developed with 3,3′,5,5′-tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA), which undergoes a color change in the presence of the antibody/allergen complexes. The absorbance (A) at 450 nm was measured using a Tecan sunrise microplate reader (Tecan, Salzburg, Austria) and the Magellan CE software after addition of 0.5 M H₂SO₄ to stop color development. The cut-off value was determined as the mean absorbance plus twice the value of the standard deviation of 20 negative controls.

The inhibitory effects of each allergen were examined using a competitive ELISA approach. For these experiments, recombinant protein was suspended in coating buffer (2 µg/ml, 50 mM carbonate buffer, pH 9.6) and added to the wells of ELISA plates. After blocking with 1% BSA in PBST for 1 h at room temperature, wells were incubated with selected serum samples (1:9; a pooled serum of 10 subjects) that had been pre-incubated overnight at 4°C with solutions containing various concentrations (0.001, 0.01, 0.1, 1.0 or 10.0 µg/ml) of recombinant proteins. IgE antibodies were detected as described above. The percentage of inhibition was calculated as (1−A_i/A₀)×100, where A_i stands for absorbance at 450 nm with an inhibitor and A₀ for the absorbance at 450 nm without an inhibitor. These assays were conducted in duplicate.

Correlations between the IgE reactivities to recombinant proteins were analyzed by Pearson's correlation using GraphPad Prism 6.0 (GrahPad, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The amino acid sequences of the six group-5 and group-21 allergens, Der f 5, Der f 21, Tyr p 5, Tyr p 21, Blo t 5 and Blo t 21, were compared. As shown in Table I, the sequence of each allergen was 36.6–55.8% identical to that of the other group-5 and -21 allergens. While Der f 5 exhibited the highest level of identity to Blo t 5 (44.4%), Der f 21 was most similar to Tyr p 5 (41.6%), Tyr p 5 was most similar to Blo t 5 (52.9%) and Tyr p 21 was most similar to Blo t 21 (55.8%). The lowest level of similarity was observed between Der f 5 and Blo t 21 (36.6%), while the highest sequence identity was observed between Tyr p 21 and Blo t 21 (55.8%) (Table I). This high level of sequence identity between allergens suggested cross-reactivity between group-5 and group-21 allergens. As depicted in Fig. 1, four amino acids that were previously found to be crucial for IgE binding, were shown to be conserved in five of the allergens: Der f 21 (E-77, D-82, E-87 and E-92), Tyr p 5 (E-78, D-83, E-88 and E-93), Tyr p 21 (E-82, D-87, E-92 and E-97) Blo t 5 (E-76, D-81, E-86 and E-91) and Blo t 21 (E-74, D-79, E-84 and E-89). Der f 5 (L-74, D-79, E-84 and E-89) was the only allergen in which the binding site was not conserved.

Recombinant Der f 5, Der f 21, Tyr p 5, Tyr p 21, Blo t 5 and Blo t 21 were inducibly expressed in E. coli. The recombinant proteins were purified from E. coli inclusion bodies by affinity chromatography using a Ni-sepharose resin. Purified proteins were then separated by 15% SDS-PAGE and visualized by Coomassie Brilliant Blue staining. As expected, the purified proteins migrated slightly slower than the 15-kDa marker, which was consistent with the predicted 17-kDa size of the allergens (Fig. 2A).

The IgE reactivity of each purified recombinant allergen was examined by ELISA using sera from 194 Korean patients who were allergic to HDM. Der f 2 was used as a positive control. While 187 of the 194 sera (96.39%) tested showed reactivity to rDer f 2, 126 (64.95%) to rDer f 5, 128 (65.98%) to rDer f 21, 59 (30.41%) to rTyr p 5, 80 (41.24%) to rTyr p 21, 60 (30.93%) to rBlo t 5 and 42 (21.65%) to rBlo t 21 (Fig. 2B). The IgE reactivity of rDer f 5 was also strongly correlated with that of rDer f 21 (r=0.733). The IgE reactivity to rTyr p 5 exhibited the highest correlation with that to rTyr p 21 (r=0.950). It also moderately correlated with those of rDer f 5 (r=0.563), rDer f 21 (r=0.613) and Blo t 5 (r=0.742). The IgE reactivity to rTyr p 21 correlated the highest with the reactivities to rDer f 5 (r=0.571), rDer f 21 (r=0.651) and Blo t 5 (r=0.704), while the reactivity to rBlo t 5 was highly correlated with those to rTyr p 5 (r=0.742) and rTyr p 21 (r=0.704). However, the IgE reactivity to rBlo t 21 exhibited a poor correlation with those to rDer f 5, rDerf 21, rTyr p 5 and rTyr p 21 (r=0.270, 0.305, 0.254, 0.349, respectively) (Fig. 3). These results demonstrated that the IgE reactivity of all six recombinant allergens exhibited at least a certain level of correlation to one another.

In order to investigate the cross-reactivity between the allergens, the present study performed an IgE inhibition on each allergen, using a competitive ELISA approach with serum pooled from 10 allergy patients (Table II). The IgE reactivities to rDer f 2, rDer f 5 and rDer f 21 were only inhibited by the selfsame allergens. rDer f 21 was found to be the strongest inhibitor of IgE reactivity to the other allergens, inhibiting 93.01% of IgE reactivity to rTyr p 5, 92.12% of that to rTyr p 21, 86.99% of that to rBlo t 5 and 70.30% of IgE reactivity to rBlo t 21, at an inhibitor concentration of 10 µg/ml (Fig. 4). The IgE reactivity to rBlo t 21 was inhibited by up to 35.27% by rDer f 5, and by up to 43.57% by rTyr p 21. rTyr p 5 inhibited 76.77% of IgE reactivity to rTyr p 21, while rTyr p 21 inhibited 86.0% of the IgE reactivity to rTyr p 5. The IgE reactivity of Blo t 5 was inhibited by up to 77.35% by rTyr p 5 and by 73.44% by rTyr p 21. In addition, rBlo t 21 inhibited the IgE reactivity to rTyr p 21 by up to 43.57%. These results suggested that each of the allergens tested exhibited a certain degree of cross-reactivity to one another; however, there was an extremely low degree of cross-reactivity between Der f 5 and the other allergens tested, which may be due to the lack of a conserved IgE binding site of Der f 5.