Serum samples were collected from HDM allergy patients in the Allergy-Asthma Clinic at Severance Hospital, Seoul, Korea. Patient consent was obtained before blood collection. Specific IgE to Dermatophagoides farinae was determined using the ImmunoCAP system (Phadia, Uppsala, Sweden). One hundred twenty-nine subjects (62 males and 67 females, mean age 34 years; range, 5 to 79 years) were enrolled in this study. Of 40 subjects with asthma, 15 were diagnosed with asthma (AS) only, 23 with AS and allergic rhinitis (AR), and 2 with AS, AR, and AD. Among 52 patients diagnosed with AR, 27 had AR only, 23 had AR with AS, and 2 had AR, AS, and AD. Of 64 individuals with AD, 58 were diagnosed with AD only and 6 were diagnosed with AD, AR, and AS. Four of these patients were also diagnosed with allergic conjunctivitis (AC). However, statistical analysis was not done for patients with AC due to the small size of this group. Subjects were assigned to the respiratory allergy group, AD group, or both allergy groups for further analysis (Fig. 1). This study was approved by the institutional review board of our institute (4-2013-0397).

Allergen extract was prepared as previously described.1011 Briefly, allergens were extracted with bicarbonate buffer from HDM bodies which were purified using saturated salt water. The extract was extensively dialyzed against distilled water and lyophilized. For the experiments, the extract was reconstituted in a phosphate buffer, aliquoted, and kept at -70℃ until use. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA).

Recombinant Der f 1 (proDer f 1) was produced as described by Yasuhara et al. with slight modification.12 Reverse transcriptase (RT)-PCR was carried out using oligonucleotide primers (forward: 5'-CTCGAGCGTCCAGCTTCAATCAAAACT-3', reverse: 5'-GCGGCCGCTTAGTGATGGTGATGGTGATGCGCGCCGCGTGATGGTG-3') to amplify Der f 1.0101, which is the predominant isoform in Korea.13 The underlined sequences corresponded to Xho I and Not I sites, and codons for 6 histidines were also incorporated in the reverse primer. PCR products were ligated into the pCR4-TOPO vector (Invitrogen Life Technologies, Carlsbad, CA, USA). To avoid hyperglycosylation, the 53rd amino acid (N-glycosylation site) was mutated from Asp to Gln using the QuikChange®II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The mutated plasmid was subcloned into the Xho I and Not I sites of the pPIC9 vector (Invitrogen). Subsequently, Sal I-linearized plasmid was transformed into GS115 cells using the PichiaEasyComp kit (Invitrogen). His+ transformants were selected on RDB plates (1.34% yeast nitrogen base without amino acids, 1 M sorbitol, 1% dextrose, 4×10^-5% biotin, and 0.005% each of L-glutamic acid, L-methionine, L-lysine, L-leucine, and L-isoleucine). A clone was selected, and cells were grown for 4 days at 220 rpm; 0.05% methanol (v/v) was added every 24 hours. Culture supernatant was harvested by centrifugation and concentrated by ammonium sulfate precipitation (50%). Precipitates were dissolved in 10 mM imidazole, 300 mM NaCl, and 50 mM sodium phosphate at pH 8.0. Recombinant proteins were purified using Ni-nitrilotriacetic (NTA) acid resin (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Mature Der f 1 was obtained by an auto-activation (cleavage of prosequence) process by dialysis against 100 mM acetate buffer (pH 4.0) at 4℃ for 48 hour.12 After this activation process, recombinant Der f 1 protein was dialyzed against 20 mM Tris-HCl at pH 8.0.

Der f 2.0102 was produced because it is the predominant isoform in Korea.13 DNA fragments were amplified by PCR (forward primer: 5'-GATCAAGTCGATGTTAAAG-3', reverse: 5'-TTATCACGGATTTTACCATG-3') using a clone ligated into a plasmid vector (pCR4-TOPO) (Invitrogen) as template, and these fragments were then subcloned into the pEXP-5NT/TOPO vector (Invitrogen). Der f 6 was amplified by RT-PCR with specific primers (forward: 5'-GATGCACGATTTCCACGC-3' reverse: 5'-TCAAACAATGTTTTTTGT-3'). PCR-products were cloned into the pEXP-5NT/TOPO vector. Der f 8 was also amplified by RT-PCR with primers (forward: 5'-ATGGCTCCAAAAACAATTCTT-3', reverse: 5'-TTATGCATATGTACCATTCCA-3'), and then cloned into the pEXP-5NT/TOPO vector. Der f 10 was PCR-amplified from the pGEM-T Easy vector (Promega) and subcloned into the pET28b expression vector (Novagen, Madison, WI, USA) as previously described.14

For production of recombinant Der f 20, PCR was performed with specific primers (forward primer: 5'-CCATGGTTGATCAAGCTGTCATCG-3', reverse primer: 5'-CTCGAGCATGGATTTTTCAATTTTAAT C-3') designed on the basis of the known cDNA sequence of arginine kinase from Dermatophagoides farinae in GenBank (accession no. AY211951). Underlined sequences correspond to Nco I and Xho I sites, respectively. Amplified PCR product was ligated into the expression vector pET28b (Novagen) after digestion with Nco I and Xho I restriction endonucleases.

All recombinant allergens except Der f 1 were expressed in Escherichia coli BL21 (DE3) and purified using Ni-agarose resin (Qiagen). Der f 2, Der f 6, and Der f 20 were isolated from inclusion bodies using 6 M urea. Der f 8 and Der f 10 were purified from soluble fractions. The concentrations of purified proteins were determined by Bradford assay, and a 10-µg aliquot of each protein was run on a 15% polyacrylamide gel containing sodium dodecyl sulfate under reducing conditions.

Levels of allergen-specific serum IgEs were measured by ELISA. Each allergen (2 µg/mL for recombinant protein and 10 µg/mL for allergen extract) in 50 mM carbonate buffer (pH 9.6) was coated onto a microtiter plate and kept at 4℃ overnight. After blocking with 3% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST), plates were incubated for 1 hour with serum samples at 1:4 dilution. Subsequently, plates were incubated with biotinylated goat anti-human IgE at 1:1,000 dilution (Vector, Burlingame, CA, USA) for 1 hour and streptavidin-peroxidase (Sigma-Aldrich, Sydney, Australia) at 1:1,000 dilution for the detection of IgE antibodies. Plates were washed three times with PBST between each step. Color was developed using 3,3'5,5'-tetramethylbenzidine substrate solution (Kirkegaard Perry Laboratories, Gaithersburg, MD, USA), and then absorbance at 450 nm was measured. The mean plus 2 SD of absorbance values of sera from non-sensitized subjects was used as the cutoff value.

Multivariate regression analysis was performed to analyze statistical significance. Correlations of IgE antibody responses between groups of patients were assessed by Pearson's χ² test and Fisher's exact test. Correlations between the numbers of subjects diagnosed with allergies according to allergy type, and the number of sensitized allergens were analyzed by ANOVA with Bonferroni correction. SPSS v18.0 was utilized, and a result was considered statistically significant when the P value was less than 0.05.

All expressed recombinant allergens showed an apparent single band with the expected molecular weight on SDS-PAGE analysis. However, Der f 2 appeared as a strong dimer along with a monomer (Fig. 2).

Subjects suffering from respiratory allergies (AR, AS, and/or AC) and/or from AD were analyzed for IgE reactivity to recombinant Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20 by ELISA (Fig. 3). As expected, most subjects had IgE responses to group 1 and 2 allergens. Der f 1- and Der f 2-specific IgEs were found in 86.4% and 89.4% of respiratory allergic patients (n = 65), respectively. Only 4.6 to 6.2% of HDM respiratory allergy patients had specific IgE to each minor allergen (Der f 6, Der f 8, Der f 10, and Der f 20). Anti-group 1 and 2 IgEs were found in 72.4% and 71.0% of AD patients (n=58), respectively. Only 5.2 to 12.1% of the AD patients had specific IgEs to these minor allergens. When all HDM allergy patients were considered, 79.1% and 79.1% of patients had IgEs specific to both group 1 and 2 major allergens, respectively, while only 6.2% to 9.3% of subjects had IgE specific to each minor allergen.

Diagnostic sensitivity according to combinations of various recombinant allergens was determined (Table 1). Der f 2 showed the highest diagnostic sensitivity in respiratory allergic patients (89.4%). The combination of 2 major allergens increased the sensitivity to 92.3%. The combination of all minor allergens with major allergens only resulted in the diagnosis of one additional subject. Approximately 84.5% of AD patients could be diagnosed as HDM allergy by the combination of 2 major allergens. Minor allergens did not increase sensitivity. In patients with both HDM-sensitized respiratory allergy and AD, neither Der f 1 (66.7%) nor Der f 2 (50.0%) was useful for diagnosis.

To investigate the usefulness of CRD for the differentiation of each HDM-related allergic disease, the relationship between sensitization profile and disease entity were examined statistically (Table 2). HDM-sensitized respiratory allergic patients had Der f 2-specific IgE more frequently than AD patients (P=0.013). The mean number of sensitized allergens tested in all enrolled subjects was 1.85, and it was not different between the HDM respiratory allergy and AD groups. There was no statistically significant relationship between the number of sensitized allergens per patient and the number of disease entities such as AS, AR, AC, or AD.