Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1

BACKGROUND: The group 1 mite allergens are the most significant indoor allergens and they belong to the papain-like cysteine protease family. To date there is only one published report on the isolation and characterization of group 1 allergens from Blomia tropicalis mites. The aims of the study are to determine the cross-reactivity between group 1 allergens and to evaluate their clinical importance in allergic patients. METHODS: The full-length Blo t 1 gene was obtained by SMART RACE cDNA amplification method using gene-specific primers. The sequence alignment was performed using LOOK followed by three-dimensional homology modeling. The cDNA was expressed in Pichia pastoris as a secretory protein. Identification of native Blo t 1 in crude mite and spent mite medium extracts was done by Western immunoblot using monoclonal antibody. Allergenicity of recombinant Blo t 1 and native Der p 1 was examined by human IgE ELISA with 80 asthmatic sera. RESULTS: The cDNA sequence consists of 1105 base pairs, including 5'- and 3'-untranslating regions, encoding an open reading frame of 330 amino acid residues. The predicted molecular weight of the deduced protein was approximately 38 kDa. Blo t 1 shared 53 and 34% nucleotide and amino acid, respectively, sequence homology with Der p 1. Native Blo t 1 was detected in both crude mite and spent mite medium extracts, and its estimated molecular weight was about 26 kDa. The recombinant Blo t 1 reacted positively with IgE in 90 and 65% of sera from asthmatic children and adults, respectively, indicating that it is a major allergen. The correlation of human IgE reactivity between Blo t 1 and Der p 1 was low in these sera. CONCLUSION: The full-length cDNA encoding group 1 Blomia tropicalis mite allergen (designated as Blo t 1) has been characterized and expressed from local mites in Singapore. This fecal allergen showed high frequency of human IgE reactivity with asthmatic sera in the tropics and there was a low correlation of IgE reactivity between Blo t 1 and Der p 1.     

Skin test reactivity to natural and recombinant Blomia and Dermatophagoides spp. allergens among mite allergic patients in the UK

BACKGROUND: Many asthmatics in tropical and subtropical areas have positive skin prick tests to both Dermatophagoides spp. and to the mite Blomia tropicalis. This may be due to recognition by IgE of cross-reactive allergens between the different mite species or because of sensitization to species-specific allergens. A 14-kDa Blomia tropicalis allergen, Blo t 5, has been cloned and shows 40% sequence homology with Der p 5. The aim of this study was to investigate reactivity to B. tropicalis in patients known to be sensitized to D. pteronyssinus and to assess allergenic activity and cross-reactivity of recombinant (r) Group 5 allergens amongst these patients, who live in the UK and who are not exposed to B. tropicalis in their homes. METHODS: Patients (n = 19) with asthma and/or rhinitis were selected based on clinical history and a positive skin prick test to D. pteronyssinus extract and were compared with non-allergic skin test negative controls (n = 10). IgE antibody responses to Blomia tropicalis, Dermatophagoides pteronyssinus, rDer p 5 and rBlo t 5 were compared by quantitative intradermal skin testing using serial 10-fold dilutions of each allergen. End point titre was the highest dilution giving an 8 x 8 mm wheal at 15 min. IgE antibodies to Blomia tropicalis, Dermatophagoides pteronyssinus, rDer p 5 and rBlo t 5 were measured using RAST, CAP and RIA, respectively. RESULTS: All 19 patients had positive skin tests to D. pteronyssinus at concentrations of 0.001 to 1 AU/ml and 10 were skin test positive to rDer p 5 at concentrations of 10-4 to 5 micro g/ml. Positive intradermal tests to Blomia tropicalis were seen in 12/19 patients at concentrations of 0.002 to 2 micro g/ml. However none of the patients had positive skin tests to rBlo t 5. Non-allergic controls were all skin test negative at the highest concentration of each allergen tested. All subjects had quantifiable specific IgE to D. pteronyssinus, but only two had IgE to B. tropicalis. IgE to Der p 5 was found in six patients, but no patients had IgE to Blo t 5. CONCLUSIONS: This study of patients naturally exposed to D. pteronyssinus but not to Blomia tropicalis, provides evidence for IgE mediated cross-reactivity between allergens produced by both mite species. The results suggest that the Group 5 allergens of D. pteronyssinus and B. tropicalis are species-specific.     

Sensitization profiles of Malaysian and Singaporean subjects to allergens from Dermatophagoides pteronyssinus and Blomia tropicalis

BACKGROUND: The house dust mites Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis (Blo t) are the most common house dust mite species in Southeast Asia. To date, there have only been a few studies on the sensitization profile of the general populations in Southeast Asia to house dust mites. The aim of this study was to determine the profiles of Der p and Blo t sensitization among Singaporean and Malaysian subjects. METHODS: Enzyme-linked immunosorbent assay was used to detect specific IgE to Der p and Blo t mite crude extracts as well as purified Der p 1, Der p 2 and Blo t 5 allergens. Sera used were from 229 Singaporean subjects (124 with rhinitis, 105 without rhinitis) and 143 Malaysian subjects (94 adults and 49 children with asthma). RESULTS: The sensitization profile of rhinitis subjects to the dust mite allergens used in this study was as follows: Blo t extract positive: 91/124 (73%); Blo t 5 positive: 62/124 (50%); Der p extract positive: 61/124 (49%); Der p 1 positive: 53/124 (43%); Der p 2 positive: 45/124 (36%). The nonrhinitis subjects' sensitization profile was as follows: Blo t extract positive: 60/105 (57%); Blo t 5 positive: 24/105 (23%); Der p extract positive: 38/105 (36%); Der p 1 positive: 14/105 (13%); Der p 2 positive: 17/105 (16%). The study of Malaysian asthmatic adults showed that 39% of them were sensitized to Der p 1, 32% to Der p 2 and 37% to Blo t 5. Among the asthmatic children, sensitization to Blo t 5, Der p 1 and Der p 2 was 90, 57 and 39%, respectively. CONCLUSION: This study clearly revealed that dual sensitization to B. tropicalis and D. pteronyssinus is common in the general populations of Singapore and Malaysia. Sensitization to Blo t 5 is more prevalent than to Der p 1 and Der p 2.     

Sensitization to Blomia tropicalis and dermatophagoides pteronyssinus-a comparative study between Singapore and Taiwan

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the predominant domestic mites species in Singapore and Taiwan. This study aims to characterize and compare the mite sensitization profiles in both countries. Skin prick tests were performed on 203 Singaporeans with Dp and Bt crude extracts. In vitro IgE and IgG4 reactivity to extracts and specific allergens (Der p 1, Der p 2 Der p 5 and Blo t 5) were determined by immunoassays. Approximately 91% of the tested Singaporeans were skin test positive for both Bt and Dp. Both populations share similar frequencies of in vitro IgE reactivity to all the allergens tested, but they differ in the pattern and magnitude of allergen sensitization. Although Der p 1, Der p 2 and Blo t5 are major sensitizing allergens in both countries, Blo t 5 is a more potent one in Singapore, probably reflecting the high level of exposure to Bt. The unique major Bt and Dp allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in both countries.     

Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus

BACKGROUND: Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens. OBJECTIVES: This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated. METHODS: Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10. RESULTS: The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal. CONCLUSION: Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.     

Allergenic differences between the domestic mites Blomia tropicalis and Dermatophagoides pteronyssinus

BACKGROUND: House dust mite allergens are the most important indoor allergens associated with asthma and rhinitis in Singapore and the tropics. Recent data to suggest that besides the Dermatophagoides spp., the domestic mite Blomia tropicalis (Bt) is also an important source of allergens in these regions. OBJECTIVE: To evaluate the degree of allergenic cross-reactivity between Bt and D. pteronyssinus (Dp). METHODS: Cross-reactivity between extracts of Bt and Dp was evaluated by fluorescent allergosorbent (FAST) inhibition studies and cross enzyme immunoelectrophoresis. Additionally, the major Dp allergens - Der p 1, Der p 2 and Der p 5, were also compared with the Bt extract by dot blot inhibition. Skin prick and intradermal end-point titration were then carried out to compare the homologous allergens of the mite species, Blo t 5 and Der p 5. RESULTS: FAST inhibition studies showed low to moderate cross-reactivity between the two dust mite extracts (maximum cross-inhibition, 60%). Native allergens studied by cross enzyme immunoelectrophoresis using mite allergic sera also showed similar results but with at least four cross-reactive IgE binding antigens. Dot blot inhibition studies using allergens of Dp, Der p 1, Der p 2, and Der p 5, showed little cross-reactivity between these allergens with components of the crude Bt extracts. Further, evaluation of a recombinant allergen of Bt, Blo t 5, showed low levels of cross-reactivity even with its homologous Dp counterpart, Der p 5. CONCLUSION: These results provide evidence that Bt allergens are distinct and have relatively low to moderate cross-reactivity with Dermatophagoides spp. allergens. Bt allergens should therefore be included in the diagnostic panel for the evaluation of allergic disorders in the tropics, and the development of new diagnostic and therapeutic strategies should include allergens of Bt.     

Correlates of sensitization to Blomia tropicalis and Dermatophagoides pteronyssinus in asthma in Barbados

BACKGROUND: Sensitivity to the mite Blomia tropicalis is related to asthma in tropical climates, but correlates of sensitivity to B. tropicalis and its relationship to Dermatophagoides pteronyssinus sensitivity have not been widely examined in families with asthma. The main objective of this study was to determine prevalence and correlates of sensitivity to these mites in families with asthma and characteristics of persons sensitized to both. METHODS: Antibodies to major antigens (Blo t 5 and Der p 1) of these mites were measured by immunochemiluminescent assay in 481 members of 29 families from Barbados ascertained through two asthmatic siblings. RESULTS: Blo t 5 sensitivity was present in 261 subjects (46%) and was associated with younger age, higher total serum IgE level, and more than a three-fold increased prevalence of asthma (42 vs. 13%). Der p 1 sensitivity was less common (27%) and showed similar associations with age, IgE, and asthma. Of the 261 subjects sensitized to Blo t 5, 116 were also sensitized to Der p 1; they were younger, had higher total and Blo t 5 specific IgE levels, and had more than twice the asthma prevalence as those sensitized to Blo t 5 alone (59 vs. 29%). Der p 1 sensitivity without Blo t 5 sensitivity was uncommon; 90% of those sensitized to Der p 1 were also sensitized to Blo t 5. Geometric mean total IgE levels were lowest in the 207 participants without any mite sensitization (102 U/ml), intermediate in 158 sensitized to either Blo t 5 OR Der p 1 (609 U/ml), and highest in 116 sensitized to both (1,869 U/ml). CONCLUSIONS: Blo t 5 is the predominant sensitizing mite allergen in these Barbadian families with correlates similar to Der p 1. Concomitant sensitization to Der p 1 appears to identify a more reactive subgroup of individuals at a higher risk of asthma.     

Prevalence of Blomia tropicalis in wheezing children in central Taiwan.

BACKGROUND AND PURPOSE: In a previous study, we found that wheezing children in rural central Taiwan had a significantly lower average sensitization rate to Dermatophagoides pteronyssinus (Der p) than those in Taipei city. We propose that Blomia tropicalis (Blo t) might be the major mite allergen in rural central Taiwan. METHODS: Using the preserved sera from our previous study, we retrospectively measured specific immunoglobulin E (IgE) antibody to Blo t and analyzed the correlation between Blo t and Der p in wheezing children in rural central Taiwan. A total of 2206 children with physician-diagnosed asthma and wheezing were enrolled and categorized among five age groups. The sensitization rate and level of specific IgE antibody to Blo t were analyzed. RESULTS: The age-specific sensitization rates and the level of specific IgE antibody to either Blo t or Der p increased progressively with increasing age, being greatest in the age group 8 to 12 years. A significant positive correlation existed between sensitization rate and age for both Blo t and Der p (p=0.001). Specific IgE antibody to Blo t was undetectable in patients younger than 1.5 years. A significant positive correlation also existed between age and anti-Blo t IgE antibody level (p<0.003). However, the allergen-specific IgE level was lower for Blo t than Der p (p<0.005) in all age groups. CONCLUSIONS: Blo t might be the major mite allergen to associated with early wheeze and atopic asthma in rural central Taiwan.     

cDNA cloning and expression of Blo t 11, the Blomia tropicalis allergen homologous to paramyosin

Blomia tropicalis is an important mite species in many parts of the world and the most predominant mite species in tropical countries. The prevalence of sensitization to this species has probably been underestimated because commercial extracts are largely unavailable. Identification and characterization of B. tropicalis allergens is an important step toward understanding the role of this species in allergic sensitization and could provide appropriate reagents for diagnostic and therapeutic procedures. This paper describes the isolation, sequence analysis, expression and allergenicity of a cDNA gene coding for a B. tropicalis allergen with homology to paramyosin, a high-molecular-weight allergen previously identified in Dermatophagoides farinae. The full-length Blo t 11 cDNA gene was isolated by cDNA library screening, 5'-rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of CLUSTAL W, CGC and BLAST program packages. The cDNA gene was expressed as a GST fusion protein in Escherichia coli and purified by affinity chromatography using the glutathione Sepharose column. Allergenicity of the rBlo t 11 was tested by human IgE dot blot immunoassay. Blo t 11 is a 3,111-bp cDNA gene with a 2,625-bp open reading frame coding for an 875-amino acid protein, exhibiting significant homology with different invertebrate paramyosins. The human IgE dot blot immunoassay showed that the rBlo t 11 reacted positively to 52% (33/63) of sera from asthmatic patients. Blo t 11 is the homolog of Der f 11 exhibiting potentially important allergenic activity.     

Identification and characterization of a novel allergen from Blomia tropicalis: Blo t 21

BACKGROUND: Allergenic components from Blomia tropicalis are important triggers of allergies in the tropics. OBJECTIVE: We sought to identify and characterize a novel allergen, Blo t 21, from B tropicalis. METHODS: Blo t 21 was initially identified from an expressed sequence tag database generated from a B tropicalis cDNA library. Allergenicity of this antigen was examined by means of skin prick testing, ELISA, and IgE immuno-dot blotting. We evaluated whether Blo t 21 and Blo t 5 were cross-reactive by using IgE inhibition ELISAs. RESULTS: Blo t 21, a 129-amino-acid protein sharing 39% identity with Blo t 5, is a product of a single-copy gene. It has an alpha-helical secondary structure and localizes to midgut and hindgut contents of B tropicalis, as well as fecal particles. Positive responses to Blo t 21 were shown in 93% (40/43) by means of ELISA and 95% (41/43) by means of skin prick testing when assayed in 43 adult patients with ongoing persistent allergic rhinitis. However, sera of 494 consecutive individuals attending outpatient allergy clinics over 1(1/2) years showed 57.9% (286/494) had positive responses to Blo t 21. Although the majority (>75%) of sensitized individuals were cosensitized to both Blo t 5 and Blo t 21, these 2 allergens had a low-to-moderate degree of cross-reactivity. CONCLUSION: Blo t 21 is a major allergen in B tropicalis that is not highly cross-reactive to Blo t 5, despite sharing some sequence and structural identity. CLINICAL IMPLICATIONS: Blo t 21, representing a new group of allergens, is an important B tropicalis allergen.     

Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients.

BACKGROUND: Blomia tropicalis has been reported to be a clinically important allergen in house dust. High prevalence of sensitization to B. tropicalis has been noted in asthmatic patients in Taiwan; however, the allergenic components and its impact on asthmatic patients remain to be clarified. OBJECTIVE: To analyze the prevalence of IgE against B. tropicalis and each allergenic component in asthmatic patients. METHODS: A series of recombinant allergenic components were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the Pharmacia CAP System and immunoblot analysis. RESULTS: A total of 131 patients were included in this study: 44% of these 131 patients were allergic to B. tropicalis, 43% of the 80 B. tropicalis-sensitive patients were allergic to Blo t 5, and 75% of the 65 Blo t 5-sensitive patients were allergic to Blo t 5 fragment 3 (Blo t 5 70-117). The sera IgE binding activity to B. tropicalis was repeatedly tested after Dermatophagoides pteronyssinus absorption, and results showed that most patients were concurrently sensitized to D. pteronyssinus and B. tropicalis. In addition, in 2 (18%) of 11 patients, the B. tropicalis sensitization was caused by the cross-reactivity of D. pteronyssinus. CONCLUSION: A high prevalence of B. tropicalis sensitization was detected in our asthmatic patients, and most of them were concurrently sensitized to D. pteronyssinus and B. tropicalis. The major allergenic component and its IgE binding fragments in Blo t 5 have been identified. These allergenic components can be used for the allergenic determination in B. tropicalis and for further immunotherapy.     

The structure of the mite allergen Blo t 1 explains the limited antibody cross‐reactivity to Der p 1

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro‐form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface‐exposed residues, suggests limited cross‐reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross‐reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.     

Association between mite allergen (Der p 1, Der f 1, Blo t 5) levels and microscopic identification of mites or skin prick test results in asthmatic subjects

BACKGROUND: Mite allergens have been involved in airway sensitization and allergic diseases. Immunoassays for the identification and quantifiction of house dust mite (HDM) allergens are useful to improve the knowledge of regional mite fauna and the remediation of mite allergens in allergic diseases. The present study analyzed the association between levels of HDM allergen and results of mite identification or skin prick test (SPT) in two different areas of Bahia, Brazil. METHODS: Forty-two asthmatic subjects from a rural area (group I; n = 21) and a slum (group II; n = 21) were evaluated through SPT with HDM allergens and had dust samples collected at their homes for mite identification and allergen measurements. RESULTS: Positive SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis allergens were observed in 42.9, 38.0 and 42.9% subjects from group I and in 47.6, 19.0 and 33.3% subjects from group II, respectively. D. pteronyssinus and B. tropicalis were identified in approximately 76 and 50% of samples from both groups, respectively. D. farinae was identified in 38.0 and 9.5% of samples from groups I and II, respectively (p < 0.005). Der p 1, Der f 1 and Blo t 5 detection were associated with mite identification (p < 0.05). Association between HDM allergen levels over 2 microg/g of dust and positive SPT occurred only with D. pteronyssinus (p < 0.0001). CONCLUSIONS: D. pteronyssinus was the most prevalent mite species in this study followed by B. tropicalis and D. farinae. Immunoassays done to measure mite allergens were associated with mite-species identification. We conclude that these three mite species must be included on panels for the diagnosis of allergic airway diseases in subjects living in such regions.     

IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1

Background:  Analysis of cross-reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross-reactivity between Ascaris and two domestic mites in the tropics.
Methods:  Sera from 24 asthmatic patients were used in ELISA and immunoblotting IgE-binding inhibition assays using Ascaris , Blomia tropicalis and Dermatophagoides pteronyssinus extracts and the recombinants Blo t 10, ABA-1 and Blo t 13 as competitors. Identification of Ascaris allergens was confirmed by mass spectrometry (LC-MS/MS).
Results:  We detected at least 12 human IgE-binding components in Ascaris extract. Blomia tropicalis and D. pteronyssinus inhibited 83.3% and 79% of IgE-binding to Ascaris , while Ascaris inhibited 58.3% and 79.3% to B. tropicalis and D. pteronyssinus respectively. Mite tropomyosin inhibited 85% of IgE-binding to Ascaris . Affinity-purified human IgE to rBlo t 10 identified an allergen of 40 kDa in Ascaris extract, further confirmed as tropomyosin by LC-MS/MS. We found no evidence of IgE cross-reactivity between rABA-1 and any allergen component in mite extracts, including rBlo t 13.
Conclusions:  There is cross-reactivity between Ascaris and mites, determined by several allergens including tropomyosin and glutathione- S -transferase. In addition to its potential impact on asthma pathogenesis, Ascaris infection and mite allergy diagnosis relying on the determination of specific IgE could be affected by this cross-reactivity. ABA-1 has no cross-reactive counterpart in mite extracts, suggesting its usefulness as a more specific marker of Ascaris infection.     

Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis

BACKGROUND: Blo t 5 is a major allergen of Blomia tropicalis and its complementary DNA (cDNA) has been expressed in both prokaryotic and eukaryotic expression systems. Although the recombinant Blo t 5 has been well characterized, relatively less is known about its native counterparts and the allergenicity comparison of the native and recombinant Blo t 5 allergens has not been reported. OBJECTIVE: The aims of this study are to characterize the native counterpart of Blo t 5, and compare the allergenicity of native and recombinant Blo t 5 by in vivo and in vitro assays. METHODS: Native Blo t 5 were purified by immuno-affinity chromatography and characterized by proteomic means. The allergenicity of the allergen was evaluated by skin prick tests, human IgE ELISA, ELISA inhibition and histamine release assays. RESULTS: Native Blo t 5 consists of at least five distinct isoforms, ranging from pI 3 to 5.5. Allergenicity assessment of recombinant and native Blo t 5 based on skin reaction, IgE-binding capacity and histamine release in allergic individuals indicated that there was a good correlation between both forms of Blo t 5 in general. However, data from IgE ELISA inhibition assay revealed the presence of additional unique IgE epitopes in native Blo t 5. CONCLUSIONS: At least five distinct isoforms of Blo t 5 have been identified. Comparative assessment of native and recombinant Blo t 5 revealed that the allergenicity of these two forms was similar but not completely identical suggesting that the various isoforms of native Blo t 5 may exhibit additional unique IgE epitopes.     

Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts

BACKGROUND: Blo t 1 is a cysteine protease-like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross-reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 1 gene with in vivo electroporation and boosted intraperitoneally with recombinant Blo t 1. mAbs against Blo t 1 were generated using a methylcellulose-based hybridoma cloning kit. The native Blo t 1 was isolated by mAb affinity purification and its allergenicity was determined by ELISA. A two-site ELISA for Blo t 1 was developed using the mAbs generated. RESULTS: A DNA-based immunization protocol induced high titre Blo t 1-specific antibodies in mice. Six stable hybridoma clones secreting mAbs recognizing the native and recombinant Blo t 1 were generated. The native Blo t 1 was affinity-purified from a B. tropicalis extract and its allergenicity was determined at 63% using a panel of Singaporean and Malaysian mite allergic patients' sera. A two-site ELISA was developed, which showed a detection limit of 10 ng/mL of Blot t 1. CONCLUSION: Six Blo t 1 mAbs were successfully generated by DNA immunization. These mAbs are useful for nBlo t 1 immunoaffinity isolation and quantitative immunoassays for Blo t 1 in mite and environmental dust extracts.     

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.     

A sensitive reverse ELISA for the measurement of specific IgE to Der p 2, a major Dermatophagoides pteronyssinus allergen.

BACKGROUND: Epidemiologic studies have shown that the presence of IgE antibodies to house dust mite and other indoor allergens is an important risk factor for asthma. OBJECTIVE: The aim of this study was to develop a reverse ELISA (rELISA) for measuring specific IgE to Der p 2, a major Dermatophagoides pteronyssinus (Dpt) allergen, as a potential tool for followup of allergen immunotherapy. METHODS: Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 was used to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Sera from 48 asthmatic patients with positive skin prick test (SPT+) to D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results were compared with a group of 41 SPT asthmatic and 30 SPT- control subjects. RESULTS: The sensitivity of the two assays for Der p 2-specific IgE was 3.9 EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISA and rELISA (r = 0.74; P < 0.0001). However, rELISA was more sensitive than was cELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Der p 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT+ asthmatic patients. CONCLUSIONS: rELISA has shown to be a sensitive and alternative method for measuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cell epitopes in allergens will provide valuable information for the design of allergen analogs and peptides for immunotherapy.     

Cloning of a group 3 allergen from Blomia tropicalis mites

BACKGROUND: Blomia tropicalis is an important mite species in the tropics and subtropical regions of the world. It is well established that the allergen from this species of mite is one of the triggering factors for allergic asthma. The isolation and characterization of allergens in this mite species is desired to provide sensitive and specific reagents for diagnostic as well as therapeutic purposes. METHODS: The SMART (Clontech Laboratories, Palo Alto, CA, USA) rapid amplification of complementary DNA ends (RACE cDNA amplification) method was used to isolate the putative Blo t 3 gene. Polymerase chain reactions (PCR) were performed in the presence of specific gene primers to obtain the full-length gene, and were confirmed by DNA sequencing. The putative gene was cloned into E. coli expression vector GST-4T-1 and expressed as a fusion protein with glutathione-S-transferase (GST). The allergenicity of the GST-Blo t 3 recombinant protein was evaluated by human IgE enzyme-linked immunoassay (ELISA) and skin pricks tests. RESULTS: The full length Blo t 3 gene had 1138 base pairs, including a 105-bp long 5' nontranslated region, an ATG start codon at positions 106-108, and a stop codon TAA at positions 904-906, with an open reading frame coding for a polypeptide of 266 amino acids. Protein analysis revealed that it was a serine protease that had a prepro-mature structure that shared high sequence homology with group 3 dust mite allergens. The predicted molecular weight of the matured protein was approximately 23.8 kD with a theoretical pI of 8.87. The frequency of IgE reactivity of the recombinant protein showed up to 50% of IgE reactivity with mite allergic subjects but IgE titer was generally low. CONCLUSION: We had isolated and fully characterized the cDNA encoding an important B. tropicalis allergen that was highly homologous to Group 3 dust mite allergens and we proposed that it should be designated as Blo t 3. Its clinical importance was implicated by the high frequency of IgE reactivity with allergic sera.     

Systemic anaphylaxis after the ingestion of pancake contaminated with the storage mite Blomia freemani.

BACKGROUND: Systemic anaphylaxis after the ingestion of mite-contaminated food has rarely been reported. OBJECTIVE: To describe an 8-year-old boy in whom systemic anaphylaxis developed shortly after the ingestion of pancakes prepared with commercial pancake flour. METHODS: The patient underwent skin prick testing for house dust mites and with uncontaminated and mite-contaminated pancake flour. Specific IgE for mites and the main ingredients of the pancake flour were also evaluated, with titers for Der p 1, Der f 1, and Blo t 5 quantitated using immunochemical methods. A sample of pancake flour was examined microscopically for mites. RESULTS: The patient had positive skin prick test results to contaminated pancake flour extract (1 g/5 mL), Dermatophagoides pteronyssinus, and Dermatophagoides farinae but a negative skin test response to uncontaminated pancake flour. The patient's serum specific IgE analysis was positive for antibodies to dust and storage mite allergens. There was no response, however, to the main ingredients of the pancake mix. Microscopic examination of the pancake flour revealed the storage mite Blomia freemani. Using an immunochemical assay, we found that the contaminated flour contained 5.4 microg/g of the allergen Blo t 5 but no Der p 1 or Der f 1. CONCLUSIONS: This patient's anaphylactic episode was the result of ingestion of the storage mite B. freemani. To our knowledge, this is the first reported systemic hypersensitivity reaction caused by this mite anywhere in the world.