Identification the Cross-Reactive or Species-Specific Allergens of Tyrophagus putrescentiae and Development Molecular Diagnostic Kits for Allergic Diseases

Mite allergens are considerable factors in the genesis of allergic diseases. The storage mite Tyrophagus putrescentiae (Tp) appears in contaminated foods and household surroundings. The current diagnostic tools for Tp allergy are mostly based on crude extracts and still contain shortcomings. This study aimed to investigate the immunoglobulin E (IgE)- responsiveness profiles of Tp-allergic patients and develop a molecular diagnostic method using recombinant allergens. Allergenic components were characterized as cross-reacting or species-specific allergens, in which the effective combinations of recombinant allergens were developed and analyzed in terms of the prediction accuracy for clinical diagnosis. Seven recombinant allergens were cloned and generated to detect the IgE responsiveness of the Tp allergy. A survey on the prevalence of mite allergy showed there were higher sensitizations with IgE responsiveness to house dust mites (HDM) (78.9–80.9%) than to storage mites Tp (35.6%). Prevalence of sensitization to Tp was higher in elderly subjects. The principal IgE-binding components of Tp were Tyr p 1, Tyr p 2 and Tyr p 3. Prediction accuracy for Tp allergy by IgE-responsiveness combination D (Tyr p 1, Tyr p 2 & Tyr p 3) was with high precision (100%). Avoiding the cross-reactivity of Dermatophagoides pteronyssinus, the prediction accuracy of IgE-responsiveness combination H+ (Tyr p 1, Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 & Tyr p 20) was suitable for Tp-specific diagnosis. Panels of Tp allergens were generated and developed a diagnostic kit able beneficial to identify IgE-mediated Tp hypersensitivity.


Introduction
There is increasing evidence that the frequency of allergic diseases such as asthma, rhinitis and dermatitis has increased worldwide over the last few decades and becomes a global public health problem [1][2][3]. The indoor inhalant allergens of mites have become the most important triggers of IgE-mediated responses and allergic diseases in genetically predisposed patients [4,5]. The most

Study Subjects
A total of 298 allergic subjects (167 males and 131 females) who attended the Clinic in the Division of Allergy, Immunology and Rheumatology at Taichung Veterans General Hospital (TCVGH) were recruited for this study. These allergic patients were diagnosed with allergies by a specialist attending physician if they had at least one of the following history: asthma, rhinitis, atopic dermatitis or eczema. The collection of blood samples from patients was approved by the Institutional Review Board, TCVGH (TCVGH IRB No. C07126). Written informed consent was obtained from each participant before being enrolled in the study. Blood samples were drawn, and sera were stored for the measurement of specific IgE to D. pteronyssinus, D. farinae and T. putrescentiae.

Preparation of Mite Crude Extracts
Two kinds of mite crude extracts were used in this study. The crude extracts of D. pteronyssinus were prepared from lyophilized whole mite bodies purchased from Allergon (Angelholm, Sweden). The T. putrescentiae mites were cultured in our laboratory with a medium consisting of yeast extract and mouse chow, then mite bodies were separated from the medium. The mite collection is described in our previous study [26]. In brief, separation of the mites from the medium was achieved by gently stirring the medium with a glass rod following overnight culture, and then the mites migrated to the cover would be collected. This collection included a large proportion of mite bodies. Frozen T. putrescentiae mites were homogenized and extracted with phosphate buffer saline (PBS, pH 7.2). Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA, USA) using bovine albumin as a standard.

Detection of IgE Responsiveness in Sera of Allergic Patients against Mite Crude Extracts
The IgE responsiveness against mite crude extracts in sera of allergic patients was determined by an enzyme-linked immunosorbent assay (ELISA) as previously described [27]. Briefly, the mite crude extracts used were coated separately onto wells of polyvinyl microtiter plates (Costar, Cambridge, MA, USA) by the addition of 100 µL of a 5-µg/mL solution of crude extract in coating buffer 0.1-mol/L NaHCO 3 , pH 8.4 for 4 h at RT. After blocking with 1% skimmed milk, it was then incubated for 1 h at room temperature [28]. Wells were washed with PBS containing 0.05% Tween-20 (Southern Biotech Association, Birmingham, AL, USA) (PBST). One hundred microliters of diluted human sera (1:5 dilution) were added for overnight incubation at 4 • C. Plates were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgE (1:1000) for 1 h at RT. After washing with PBST three times, the bound enzyme substrates were detected with 3, 3', 5, 5'-tetramethylbenzidine (TMB) substrate (Invitrogen, Carlsbad, CA, USA). The reactions were stopped with 50 µL 1-N H 2 SO 4 after 15 min, and the optical density was measured at 450 nm in a multiscan spectrophotometer (Sunrise, TECAN, Mennendorf, Switzerland). The cutoff value was mean ± 2x standard deviation as 0.23 ± 0.02 unit of the sera of 10 healthy volunteers who have no history of allergic symptoms, and samples exceeding 0.25 unit were considered as positive.

Preparation, Expression and Purification Recombinant Allergens of T. putrescentiae
The recombinant allergens (rTyr p 1, rTyr p 2, rTyr p 3, Tyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20) of T. putrescentiae were prepared and used to analyze the frequency of IgE reactivity by sera from the allergic subjects. Total RNA of mites was isolated with Trizol reagent, and cDNA was synthesized with primer dT, MMLV reverse transcriptase (Gibco-BRL, New York, NY, USA). The recombinant proteins of T. putrescentiae allergenic components were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from cDNA and subcloned into the expression vector. The specific primers were designed based on the accession number in the National Center for Biotechnology Information or in our previous study finding. The program of PCR was carried out at 94 • C for 10 s, 55 • C for 20 s and 72 • C for 3 min for 35 cycles. The PCR products were purified, ligated into a pQE30 vector and transformed in Escherichia coil-M15 strain competent cells. Transformants were selected by kanamycin (25 µg/mL) and ampicillin (100 µg/mL). Expressions of recombinant allergens were performed according to the methods described in the QIA Expressionist TM Kit directions (Qiagen, Hilden, Germany). The recombinant allergens were expressed as a 6x His-tagged protein using 1-mM isopropyl-β-D-thiogalactopyranoside (Promega, Madison, WI, USA) induction. The proteins were purified by nickel-nitrilotriacetic acid agarose metal affinity column chromatography under native conditions.

Identification of T. putrescentiae-Specific Allergen by IgE Absorption with D. pteronyssinus
Because of the cross-reactivity between different mite species, the patients' sera were pre-absorbed with D. pteronyssinus then to evaluate the IgE responsiveness for identification the species-specific components of T. putrescentiae. For the IgE absorption, serum samples were selected from 106 allergic subjects who had IgE antibodies against T. putrescentiae crude extracts using ELISA. Each serum was preincubated with 50 µL of D. pteronyssinus crude extracts (50 µg/mL) and left overnight at 4 • C. After the preabsorption, the subsequent procedures for determining IgE responsiveness to T. putrescentiae by ELISA were the same as those described previously.

The Frequency of IgE Responsiveness to T. putrescentiae Recombinant Allergens
Recombinant allergens of T. putrescentiae were prepared and used to investigate the frequency of IgE responsiveness. The IgE antibodies in serum samples were detected by ELISA. The recombinant allergens (rTyr p 1, rTyr p 2, rTyr p 3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20) were coated separately onto wells of polyvinyl microtiter plates (Costar, Cambridge, Mass., USA) by the addition of 100 µL of a 5-µg/mL solution in 0.1 MNaHCO 3 (pH 8.4) for 4 h at RT. After blocking with 1% skimmed milk, it was then incubated for 1 h at RT. Wells were washed with PBS containing 0.05% Tween-20 (Southern Biotech Association, Birmingham, Ala., USA)(PBST). The ratio 1:5 dilution of serum from each allergic patient was added in 100 µL PBS. Wells were washed again with PBST, peroxidase-conjugated anti-human IgE (1:1000 dilute in PBST) was added to the wells and incubated for 2 h at RT. After washing with PBST, the bound enzyme substrates were detected with TMB substrate (Invitrogen, USA). The reaction was stopped with 50 µL 1-N H 2 SO 4 after 15 min, and the optical density was measured at 450 nm in a multiscan spectrophotometer (Sunrise, TECAN, Mennendorf, Switzerland). The cutoff value was mean ± 2x standard deviation as 0.23 ± 0.02 unit of the sera of 10 healthy volunteers who have no history of allergic symptoms, and samples exceeding 0.25 unit were considered as positive. The IgE-binding frequency of T. putrescentiae allergens among allergy patients was analyzed and compared after D. pteronyssinus absorption. Each serum was pre-incubated with 50 µL of D. pteronyssinus crude extracts (50 µg/mL) and left overnight at 4 • C. After the pre-absorption, the subsequent procedures for determining IgE responsiveness to T. putrescentiae allergens by ELISA were the same as those described previously.

Establish the Detection Combinations of IgE Responsiveness for the Diagnosis of T. putrescentiae Allergy
A total of seven allergenic components of T. putrescentiae were cloned and the recombinant allergens were generated for detection combinations of IgE responsiveness in sera from allergic patients. The higher frequency with IgE responsiveness of allergenic components are picked out, the higher sensitivity of detection combination for T. putrescentiae can be obtained. Since the cross-reactivity of T. putrescentiae and D. pteronyssinus were common in clinical practice. Sera from allergic patients with the specificity of T. putrescentiae allergy were confirmed after the absorption of D. pteronyssinus. Only those detection combinations with both high sensitivity and specificity will be used for clinical application in the future.

Statistical Analysis
Differences between the middle-age and elderly age population of the T. putrescentiae allergy were analyzed by the nonparametric alternative to the unpaired two-sample t-test. The p-values of differences less than 0.05 were considered statistically significant.

Results
3.1. The Prevalence of Sensitization to D. pteronyssinus, D. farinae and T. putrescentiae The sensitization to D. pteronyssinus, D. farinae and T. putrescentiae of allergic patients was determined by ELISA with mite crude extracts. The results showed that the IgE reaction with positive responses of allergic subjects were 80.9% (241/298) to D. pteronyssinus, 78.9% (235/298) to D. farinae and 35.6% (106/298) to T. putrescentiae (shown in Figure 1A). There were more subjects sensitized to D. pteronyssinus or D. farinae than to T. putrescentiae. It is a possible phenomenon that multiple exposures and cosensitization to these mite allergens in nature causing free IgE filled in the sera of these allergic patients. The overlapping illustration of sensitization prevalence to these mites of these subjects, the percentage of positive IgE reactions to the dust mite (D. pteronyssinus and D. farinae) and storage mite T. putrescentiae were 33.6% (100/298) ( Figure 1B). The prevalence of sensitization to D. pteronyssinus and D. farinae, but none to T. putrescentiae were 45.0% (134/298) ( Figure 1B). There were 16.8% (50/298) of these subjects without IgE response against these species of mites. It seemed that most of the allergic patients from the Taichung area had higher sensitization with positive IgE responses to house dust mites (78.9-80.9%) than to storage mite T. putrescentiae (35.6%).

Differences of Mite Sensitization between Elderly and Young Adult Subjects
Differences in sensitization to D. pteronyssinus and T. putrescentiae were compared between elderly and young adult subjects. The results showed there were 84.1% (116/138) allergic subject sensitive to D. pteronyssinus and 81.2% (112/138) sensitive to D. farinae in the young adult group; there was a slightly higher prevalence of D. pteronyssinus sensitivity in the young adult group than in the elderly group ( Figure 2). Additionally, the analysis data showed there were 44.4% (71/160) elderly subjects sensitive to T. putrescentiae while there were only 25.4% (35/138) young adult subjects allergic to T. putrescentiae. The prevalence of sensitization to D. pteronyssinus or D. farinae was slightly higher in the young adult subjects, while the prevalence of sensitization to T. putrescentiae was significantly higher in elderly subjects than in young adult subjects (44.4% vs. 25.4%) (p < 0.05).

Molecular Cloning and Purification of Recombinant Allergens from T. putrescentiae
The RNA of T. putrescentiae was isolated from alive mite bodies and the complementary DNA (cDNA) synthesized via reverse transcription [28]. The cDNA encoding for T. putrescentiae allergens was amplified by RT-PCR and subcloned into expression vectors. A total of seven allergenic components were expressed and purified by His-tag affinity column, including rTyr p 1, rTyr p 2, rTyr p 3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20. The characteristics including biologic function, molecular weight and accession No. of allergens from T. putrescentiae we used in this study is listed in Table 1. The protein profiles of T. putrescentiae recombinant allergens on SDS-PAGE showed that the rTyr p 2 approximately at 16 kDa, the rTyr p 1, rTyr p 3, rTyr p 7 and rTyr p 8 approximately at 24-26 kDa and rTyr p 10 & rTyr p 20 approximately at 37-40 kDa (shown in Figure 3). The recombinant allergens from T. putrescentiae were prepared for the subsequent identification of IgE-binding frequency by the ELISA. Diagnostics 2020, 10,

Differences of Mite Sensitization between Elderly and Young Adult Subjects
Differences in sensitization to D. pteronyssinus and T. putrescentiae were compared between elderly and young adult subjects. The results showed there were 84.1% (116/138) allergic subject sensitive to D. pteronyssinus and 81.2% (112/138) sensitive to D. farinae in the young adult group; there was a slightly higher prevalence of D. pteronyssinus sensitivity in the young adult group than in the elderly group ( Figure 2). Additionally, the analysis data showed there were 44.4% (71/160) elderly subjects sensitive to T. putrescentiae while there were only 25.4% (35/138) young adult subjects allergic to T. putrescentiae.  The prevalence of sensitization to D. pteronyssinus or D. farinae was slightly higher in the young adult subjects, while the prevalence of sensitization to T. putrescentiae was significantly higher in elderly subjects than in young adult subjects (44.4% vs. 25.4%) (p < 0.05).

Molecular Cloning and Purification of Recombinant Allergens from T. putrescentiae
The RNA of T. putrescentiae was isolated from alive mite bodies and the complementary DNA (cDNA) synthesized via reverse transcription [28]. The cDNA encoding for T. putrescentiae allergens was amplified by RT-PCR and subcloned into expression vectors. A total of seven allergenic components were expressed and purified by His-tag affinity column, including rTyr p 1, rTyr p 2, rTyr p 3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20. The characteristics including biologic function, molecular weight and accession No. of allergens from T. putrescentiae we used in this study is listed in Table 1. The protein profiles of T. putrescentiae recombinant allergens on SDS-PAGE showed that the rTyr p 2 approximately at 16 kDa, the rTyr p 1, rTyr p 3, rTyr p 7 and rTyr p 8 approximately at 24-26 kDa and rTyr p 10 & rTyr p 20 approximately at 37-40 kDa (shown in Figure 3). The recombinant allergens from T. putrescentiae were prepared for the subsequent identification of IgE-binding frequency by the ELISA.

The Frequency of IgE Binding to Each Allergenic Components of T. putrescentiae before and after D. pteronyssinus Absorption
The frequency of IgE binding to the allergen can provide important information for the diagnosis of clinical allergy. A total of 106 sera with ELISA-positive reactions to T. putrescentiae from the allergic subjects were selected for the identification of the major allergenic components by IgE immuno-blotting. The sera from T. putrescentiae sensitive subjects were selected for the measurement of IgE responsiveness to allergenic components before and after D. pteronyssinus absorption. The frequencies of IgE responsiveness to these allergens were shown as follows: For further confirmation of cross-reactivity between D. pteronyssinus, the T. putrescentiae-sensitive sera were selected for performing the IgE immunoblot inhibition with D. pteronyssinus absorption. After D. pteronyssinus absorption, the IgE-binding frequency of Tyr p 1, Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 and Tyr p 20 were decreased as 51.9%, 39.6%, 41.5%, 32.1%, 17.9%, 5.7% and 17.9% (Figure 4). The results showed that the IgE-binding frequencies of T. putrescentiae allergenic components were decreased, suggesting that there were different levels of cross-reactivity between T. putrescentiae and D. pteronyssinus. The decreased levels of IgE-binding were higher in Tyr p 8, Tyr p 10 and Tyr p 20 (three-fold decreased), following by Tyr p 2, (two-fold decreased). The level of cross-reactivity between T. putrescentiae and D. pteronyssinus was trivial in Tyr p 1, Tyr p 3 and Tyr p 7. Results showed the highest level of cross-reactivity between T. putrescentiae and D. pteronyssinus were Group 10 allergens, the following were Group 20 and Group 8 allergens. The protein sequences of these allergens were compared between T. putrescentiae and D. pteronyssinus by Basic Local Alignment Search Tool (BLAST), it showed the sequence similarities were over 80% in Group 8, Group 10 and Group 20 allergens (Supplementary Table S1). The lower similarities of protein sequences were, respectively in Group 1, Group 3 and Group 7. The analysis of sequence similarity confirmed that it was indeed consistent with the experimental demonstration of cross-reactivity performed by IgE immunoblot inhibition.     Table S1). The lower similarities of protein sequences were, respectively in Group 1, Group 3 and Group 7. The analysis of sequence similarity confirmed that it was indeed consistent with the experimental demonstration of cross-reactivity performed by IgE immunoblot inhibition.

The Frequency of IgE-Binding in T. putrescentiae Allergenic Components in the Different Age Group before and after D. pteronyssinus Absorption
The frequencies of IgE-binding to T. putrescentiae allergenic components were 44.4% (71/160) in the elderly age group (over or equal 65 years old) and 25.4% (35/138)

The Frequency of IgE-Binding in T. putrescentiae Allergenic Components in the Different Age Group before and after D. pteronyssinus Absorption
The frequencies of IgE-binding to T. putrescentiae allergenic components were 44.4% (71/160) in the elderly age group (over or equal 65 years old) and 25.4% (35/138) in the middle-age group (less 40 years old). For further identification of the allergy condition in different age groups, the sera with positive IgE responsiveness to T. putrescentiae were selected for the measurement of responsiveness to each T. putrescentiae allergenic components before and after D. pteronyssinus absorption. Before the D. pteronyssinus absorption, the frequencies of IgE-binding to each T. putrescentiae allergenic components were shown as follows: Tyr p 1 (63.6%), Tyr p 2 (78.9%), Tyr p 3 (43.7%), Tyr p 7 (39.4%), Tyr p 8 (43.7%), Tyr p 10 (12.7%) and Tyr p 20 (47.9%) in the elderly age group, respectively ( Figure 5). In the middle-age group, the frequencies of IgE-binding to allergens were shown as follows: Tyr p 1 (57.1%), Tyr p 2 (80%), Tyr p 3 (60%), Tyr p 7 (34.3%), Tyr p 8 (45.7%), Tyr p 10 (31.4%) and Tyr p 20 (48.6%), respectively ( Figure 5). Before D. pteronyssinus absorption, the frequencies of IgE-binding to the allergenic components of T. putrescentiae in the middle-age group were higher than the elderly age group, except Tyr p 1 and Tyr p 7 ( Figure 5).
Before D. pteronyssinus absorption, the major allergenic components of T. putrescentiae in the middle-age group with age under 40 years old were Tyr p 1 (57.1%), Tyr p 2 (80%) and Tyr p 3 (60%). After the D. pteronyssinus absorption, the frequencies of IgE-binding to Tyr p 1 (57.7% vs. 40%), Tyr p 2 (49.3% vs. 20%), Tyr p 7 (35.2% vs. 25.7%) and Tyr p 8 (19.7% vs. 14.3%) in the elderly age group were higher than in middle-age group. The data of statistical analysis showed the frequencies of IgE-binding were significantly reduced after the D. pteronyssinus absorption in the Group 10 and Group 8 allergens no matter in the elderly age group or middle-age group (p < 0.01; Figure 5). The frequencies of IgE-binding were significantly reduced after the D. pteronyssinus absorption in the Group 2 allergen in the middle-age group (p < 0.01; Figure 5). There were significant differences between the before and after D. pteronyssinus absorption in Group 1 and Group 20 allergens in the middle-age group (p < 0.05; Figure 5). Furthermore, an obvious decrease was observed after the D. pteronyssinus absorption in Group 2 allergen in the elderly age group (p < 0.05; Figure 5). There were higher IgE-binding frequencies of T. putrescentiae specific allergens in the elderly age group. These results suggested that majority sensitization of D. pteronyssinus was in the middle-age group and the majority sensitization of T. putrescentiae was in the elderly age group. Diagnostics 2020, 10

T. putrescentiae Allergy Diagnosis Using Several Combinations of IgE-Responsiveness Prevalence to Allergenic Components
The identification of sensitization to storage mite T. putrescentiae is important and essential in clinical diagnosis. Crude extracts based diagnostic methods for identifying mite allergy and sensitization, such as skin prick test, provocation test and extract serology, remain some disadvantages in clinical practice. In this study, we investigated whether T. putrescentiae allergenic component combinations of IgE responsiveness can provide more effective and precise methods for clinical diagnosis. Which type of combinations of the IgE responsiveness is the highest precision of prediction

T. putrescentiae Allergy Diagnosis Using Several Combinations of IgE-Responsiveness Prevalence to Allergenic Components
The identification of sensitization to storage mite T. putrescentiae is important and essential in clinical diagnosis. Crude extracts based diagnostic methods for identifying mite allergy and sensitization, such as skin prick test, provocation test and extract serology, remain some disadvantages in clinical practice. In this study, we investigated whether T. putrescentiae allergenic component combinations of IgE responsiveness can provide more effective and precise methods for clinical diagnosis. Which type of combinations of the IgE responsiveness is the highest precision of prediction accuracy was estimated. The IgE responsiveness to T. putrescentiae allergenic components were chosen to composite five combinations,

including combination A (Tyr p 1 & Tyr p 2), combination B (Tyr p 2 & Tyr p 3), combination C (Tyr p 3 & Tyr p 7), combination D (Tyr p 1, Tyr p 2 & Tyr p 3) and combination E (Tyr p 2, Tyr p 3 & Tyr p 7)
to determine the prediction accuracy. All possible permutations of allergen IgE responsiveness were analyzed to find out the most accurate combination set. Because of the size of the table, only a few combinations of five sets with high predictive value over 80% of T. putrescentiae allergy are shown in Table 2. The results showed that the prediction accuracy for T. putrescentiae allergy by IgE-responsiveness combination A, B and C could be reached up to eighty percent (84.0-88.7) ( Table 2). The prediction accuracy for T. putrescentiae allergy by IgE-responsiveness combination D (Tyr p 1, Tyr p 2 & Tyr p 3) and E (Tyr p 2, Tyr p 3 & Tyr p 7) could be achieved to 100% (Table 2). It seems these major allergens are more suitable for diagnostic agents. The IgE inhibition between T. putrescentiae and D. pteronyssinus was used to evaluate the cross-reactivity and to identify different components belongs to cross-reacting allergens or species-specific allergens. In order to consider the influence of cross-reactivity from house dust mite, the IgE adsorption experiment was performed. The data of IgE-responsiveness combinations to species-specific allergens with high predictive value over 55% of T. putrescentiae allergy are shown in Table 3 (Table 3). Moreover, the prediction accuracy of IgE-responsiveness combination H+ (Tyr p 1, Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 & Tyr p 20) could be achieved to 100%, it showed this combination H+ is more suitable for T. putrescentiae-specific diagnosis. Table 3. Diagnostic combination for T. putrescentiae allergy after Dp absorption.

Discussion
Inhaled allergens are considerable factors in the genesis of allergic diseases. The ubiquitous presence of T. putrescentiae in urban and rural settings is an important occupational hazard in agricultural environments or a common allergy-provocation factor in household surroundings [29]. In view of existence abundance and high allergenicity, the house dust mite D. pteronyssinus, D. farinae and storage mite T. putrescentiae are at high levels of infestation and associated with airway hypersensitivity for the development of allergic diseases [30,31]. The prominent appearance and abundance of mites may be influenced by the warm and humid climate in Taiwan. D. pteronyssinus is the major cause of allergy in more than 80% of allergic patients with bronchial asthma in Taiwan [8,32], and the major allergens have been identified as Der p 1, Der p 2 and Der p 3 [8]. Most of the allergic patients with mite allergy are almost allergic to both species of mites, D. pteronyssinus and D. farinae, which are the predominant species and coexist in most geographical regions [33]. The phenomenon can be speculated that it may be cosensitized to D. pteronyssinus and D. farinae in the household surroundings or possessed high levels of immunologic cross-reactivity between their allergenic components [33]. There is a high prevalence of T. putrescentiae sensitization in the general adult population in both Europe [34] and Asia [35]. The prevalence of T. putrescentiae sensitization in the United States is also very important that there is 34.3% serum sample sensitized both to T. putrescentiae and D. pteronyssinus [36]. The storage mite T. putrescentiae has often been identified in the house dust samples from South America [37]. T. putrescentiae is the most prevalent species of storage mite in the dry-stored food products from house and retail stores in Egypt of Africa [38]. In Spain, where the climate is rainy and temperate, D. pteronyssinus-allergic patients also show a very high prevalence (73-83%) of storage mite sensitization [34]. There, 52% of adult individuals over the age of 50 are sensitive to T. putrescentiae, which is much higher than D. pteronyssinus (22%) in the same age group. Similar findings have been reported in Korea. T. putrescentiae is the third most common mite in the indoor environment and around one-third of individuals are allergic to it [35]. Despite the high prevalence of T. putrescentiae sensitization in the general adult population, especially in individuals over 50 years old, there are few reports on its clinical significance in elderly populations. It is of interest to evaluate the allergenicity, prevalence of hypersensitivity and clinical relevance of T. putrescentiae sensitization in elderly populations, this phenomenon implicates that D. pteronyssinus and T. putrescentiae may have different sensitization exposure.
Recently, storage mites have been reported to be clinically important allergenic components of dust samples throughout the world, supporting their clinical significance [39]. In our previous report, we demonstrated that 63% of asthmatic patients had concomitant sensitization to storage mite B. tropicalis and house dust mite D. pteronyssinus by skin tests and the molecular structure and cross-reactivity between Blo t 5 and Der p 5 were estimated at 33% to 43% by IgE inhibition [40].
This suggests that several IgE-binding proteins of storage mites are cross-reactive components, which can be inhibited by D. pteronyssinus, but the others are species-specific allergens [40]. In our previous study, we had investigated the relationship between T. putrescentiae and D. pteronyssinus allergy in allergic rhinitis individuals by using skin prick tests, basophil histamine release assays in vitro and specific IgE measurements [18]. The reactivity between T. putrescentiae and D. pteronyssinus was investigated and results showed that the major cross-reacting allergen was Group 2 allergens and species-specific allergen was Group 3 allergens [27].
It is also a paramount challenge to develop a small amount of blood for the diagnosis of mite allergy. The study can provide a solution to detect many allergens with a small amount of blood to identify which allergenic components of T. putrescentiae to cause immune responses for developing allergic diseases. For allergy diagnosis, the purity and the representativeness of the allergens should also be considered. Since the diagnosis of T. putrescentiae allergy is still based on the crude extracts and some of the allergenic components which been found or obtained. Some of these allergenic components possess enzymatic activities causing the instability of allergen preparation which results in IgE-binding activity varied and difficult to make an accurate diagnosis. That is the explanation why the divergences of allergen contents had been mentioned that the levels of both Der p 1 and Der p 2 in D. pteronyssinus extract from the different company reveal variability and the proportion variation of Der p 1 and Der p 2 among different extracts are likely to influence their biologic effectiveness [41]. Therefore, the recombinant allergens which reserved immunological activity of the natural allergen can be used for component-resolved-diagnosis (CRD) of the patient's allergen sensitization profiles of Type I allergy, whereas avoiding disadvantages that are difficult to standardize allergen contents and contain additional undefined nonallergenic components in the crude extracts [42]. The CRD based on recombinant allergens can use to establish IgE sensitization profiles in the polysensitized patients and to realize cosensitization and cross-reactivity of the allergen components [43]. The potential association between allergen components causing allergic symptoms can be further understood using CRD [43]. CRD enables testing for a specific IgE against multiple allergen components, it can be very useful in the diagnosis of anaphylaxis caused by unknown allergens [44]. CRD can also be applied to the diagnosis of food allergy, pollen allergy, occupational asthma, illicit drug hypersensitivity and utility in deciding immunotherapy [44]. The ImmunoCAP © ISAC allergen-microarray (Phadia, Uppsala, Sweden) for the determination of specific IgE against recombinant or purified allergen components has good analytical performance when compared with the traditional method of ImmunoCAP© 250 [45]. In our study, recombinant allergens of Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 and Tyr p 20 were cloned and generated to detect specific IgE in the serum of T. putrescentiae sensitized patients. The prediction accuracy for T. putrescentiae allergy by IgE-responsiveness combination D (Tyr p 1, Tyr p 2 & Tyr p 3) and E (Tyr p 2, Tyr p 3 & Tyr p 7) could be achieved to 100%. It seems the prediction combinations of these recombinant allergens from major allergens are suitable for diagnostic agents. Recombinant allergens have provided us with high accuracy and efficiency that can improve the diagnosis of allergy.
Childhood asthma and adult-onset asthma are a globally significant immune-disorder disease with major public health consequences known to share many of the same causes and triggers [46]. Allergic sensitizations and environmental exposures including mite allergens are associated with asthma severity in children and adolescents [46]. The component-specific IgE responses associated with diverse risk of asthma existence, continuance and severity have been identified using component-resolved diagnostics rather than standard skin tests and blood tests to the whole allergen extracts in children [47]. The evolution of IgE responses to multiple allergen components throughout allergy pathogenesis described in this study, which may facilitate the development of better diagnostic and prognostic biomarkers for allergic diseases.
It is feasible to generate a panel of T. putrescentiae allergenic components and develop an immunoassay kit to identify the IgE-mediated hypersensitivity of T. putrescentiae. A patent for the diagnostic kit (patent No. I 467177) for T. putrescentiae allergy including the allergens of rTyr p2, rTyr p3, rTyr p 7, rTyr p 8, rTyr p 10 and rTyr p 20 has been registered and certified in Taiwan, the Republic of China (expired time 04/22/2032). This is based on a combination with high-frequency IgE-binding component of Tyr p 2 and the high species-specific allergenic components of Tyr p 3 & Tyr p 7, a refined microarray panel of allergens. The development of diagnostic kits can be identified T. putrescentiae IgE-mediated hypersensitivity more specifically the detectable allergens would broaden involved more species-specific allergens in T. putrescentiae, which avoid the cross-reactive allergen of D. pteronyssinus whenever possible.
Tyrophagus putrescentiae is one of the most common storage mites found in the storage rooms, kitchen and frequently contaminated in the mushrooms, farina and pet food [48]. There are more than 80% of dust samples contained mites, including house dust mites (D. pteronyssinus and D. farinae) and storage mites (T. putrescentiae) be identified in the dust samples [40,49]. Antigens of storage mite are predominantly found in the kitchens and antigens of house dust mite are mainly detected in the bedrooms, especially the T. putrescentiae is the most prevalent mite in all room types [49]. The mite bodies and feces of T. putrescentiae can be accumulated in the contaminated foods or residential surroundings causing food allergy and airway hypersensitivity [48,50]. Although there are at least 36 allergenic components reported in D. pteronyssinus, in contrast only a few allergens of T. putrescentiae been identified and characterized. In the currently identified allergens of T. putrescentiae, there are some major allergens such as Tyr p 1, Tyr p 2 and Tyr p 3 with higher than 50% IgE-binding activity in the T. putrescentiae sensitized patients caused hypersensitivities.
The seven allergenic components were used to identify the IgE-binding activity and to confirm the IgE immunoblot inhibition, it showed that all of the IgE-binding activities decreased after D. pteronyssinus absorption indicating certain levels of cross-reactivity between T. putrescentiae and D. pteronyssinus. In this study, the highest level of cross-reactivity allergen between T. putrescentiae and D. pteronyssinus was the Group 10 mite allergen. The Group 10 mite allergens are involved in muscle contraction in invertebrates and processed high levels of cross-reactivity between mites, shrimp and insects in seafood-allergic patients, referred to as pan-allergens [51]. The cross-reactivity between T. putrescentiae and D. pteronyssinus was confirmed after the IgE absorption and protein sequence comparison; it showed the most cross-reactive allergens were Group 10, Group 20 and Group 8. Another highly conserved component had been identified as Group 2 allergen, similar findings demonstrated that the IgE-binding activity of Tyr p 2 could be extremely absorbed by Der p 2 and the Group 2 allergens are the major cross-reactive allergen of T. putrescentiae and D. pteronyssinus [18]. The Der p 2 is a lipid-binding protein [52], which binds lipopolysaccharides (LPS) in a style similar to binding by myeloid differentiation protein-2 (MD-2) onto Toll-like receptor-4 (TLR-4) [53]. The allergenicity of Group 2 mite allergens may be heightened by a similar structure interaction [54], therefore about 80-100% of mite-allergic patients are primarily sensitized to Der p 2 [8], the results as our research found that about 80% were sensitized to Tyr p 2 in the T. putrescentiae-allergic patients. Most the surface residues are conserved leading to similar epitopes forming the structural basis of cross-reactivity between Group 2 allergens of dust mites [55]. Not all of the allergenic components possessed high levels of cross-reactivity between T. putrescentiae and D. pteronyssinus, such as Tyr p 3 and Tyr p 7, the reduction of IgE-binding activity after D. pteronyssinus absorption were trivial. The similar finding showed there are poor correlations of serum specific IgE to Tyr p 3 and Der p 3, which with poor cross-reactivity between them and the inhibition degree by Group 3 allergens (Tyr p 3) is lower than by Group 2 allergens (Tyr p 2) in the T. putrescentiae-allergic patients [18]. Overall, it suggests Tyr p 3 is a major non-cross-reactive component belongs to a T. putrescentiae specific allergen and may serve as a suitable candidate for a differential diagnostic marker for T. putrescentiae hypersensitivity.
The main limitations of this study are related to the usage of allergen groups and the recruitment of T. putrescentiae-allergic patients. Not all of the identified allergens of T. putrescentiae were used for the development of diagnostic kits in this study. Although not all allergenic components are included, most major allergens are included for diagnosis and the prediction accuracy is close to 100%. In this study, just 106 T. putrescentiae-allergic patients were used to check the accuracy of diagnostic kits. Actually, a total of 298 allergic patients were recruited in the Clinic of the Division of Allergy, only about 1/3 of allergic patients were allergic to T. putrescentiae with IgE positive responses. This is can be contributed to more than 80% of the dust samples contained house dust mites including D. pteronyssinus and D. farinae in Taiwan [56]. Most of these allergic patients were exposed to them by contact with mattresses or carpets in the household environment then sensitized to house dust mites of D. pteronyssinus and D. farinae [57]. Because of their abundance and high allergenicity of D. pteronyssinus and D. farinae, the major predominant mites that elicit positive IgE responses and provoke airway hypersensitivity in Taiwan [48]. Another limitation was that we do not evaluate the associations between the levels of other antibodies except the IgE and severity of the allergy. The pathogenesis and severities of allergic diseases are actually related to many factors such as inflammatory mediators (cytokines or chemokines), granulocytes (mast cells, basophils and eosinophils), T cell differentiation and airway remodeling [58]. In this study, we focused more on the IgE-mediated hypersensitivity and improved the accuracy of the diagnostic combination of allergenic components.

Conclusions
The storage mite T. putrescentiae is an important occupational hazard in agricultural environments, accident ingestion of mite-contaminated foods and ubiquitous presence in urban or rural settings, it is also a common allergy-provocation factor in household surroundings. So, the diagnosis method of T. putrescentiae allergy with convenience and accuracy is necessary for clinical treatment. The allergy diagnosis based on the crude extracts still contains some disadvantages and insufficiencies. The recombinant allergens can be used for understanding the sensitization profiles of allergic patients. In this study, we found out prediction accuracy of IgE responsiveness for T. putrescentiae with allergen combination (Tyr p 1, Tyr p 2 and Tyr p 3) could be achieved to high precision (100%). Avoiding the allergen cross-reactivity from predominant mite D. pteronyssinus performed by IgE absorption, the allergenic components of Tyr p 3 and Tyr p 7 have been identified as species-specific allergens. The IgE responsiveness to species-specific allergens is more suitable for the diagnosis of T. putrescentiae allergy focusing on IgE-mediated hypersensitivity.

Conflicts of Interest:
The authors declared that they have no conflicts of interest.