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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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Background: Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens., Objective: To evaluate the differences in the IgE response against two Blo t 12 isoallergens., Methods: The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens., Results: The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes., Conclusion: In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy.

Background: There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations., Objective: We aimed to examine the allergenic specificity of IgE binding in sera from house dust mite (HDM)-atopic subjects in a tropical Australian Aboriginal community., Methods: Sera shown to contain IgE antibodies to an HDM extract of Dermatophagoides pteronyssinus were examined for IgE binding to a panel of nine purified HDM allergens from this mite species by quantitative microtitre assays. IgG antibody binding (IgG1 and IgG4) was also measured., Results: The IgE-binding activity in the sera from the Aboriginal community was not directed to the expected major groups 1 and 2 HDM allergens but instead to the group 4 amylase allergen. There was also little IgE binding to the potentially cross-reactive tropomyosin (Der p 10) or arginine kinase (Der p 20) allergens. The IgG4 antibody was rarely detected and limited to the Der p 4 allergen. IgG1 antibody binding was frequently measured to all the allergens regardless of an individual's atopic status, whereas in urban communities it is restricted to the major allergens and to atopic subjects., Conclusion: The high IgE anti-HDM response of Australian Aboriginals predominantly bound Der p 4 and not the Der p 1 and 2 allergens, showing a distinctive allergy that could affect the disease outcome and diagnosis.

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.

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.

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.

Background: The identification of immunodominant peptides containing the IgE and IgG epitopes on allergen molecules is an important step in understanding the interaction of the allergen with the immune system and, thus, essential for the development of effective immunotherapeutic and diagnostic reagents. The present study aimed to map the IgE and IgG immunodominant peptides of Blomia tropicalis (Bt) allergen Blo t 11, a high molecular weight allergen homologous to paramyosin, exhibiting important allergenic activity., Methods: Eleven overlapping fragments of Blo t 11 cDNA gene were expressed as glutathione s-transferase (GST) fusion peptides, which were affinity-purified using the glutathione-Sepharose column. Human IgE and IgG immunodominant peptides were determined by dot blot immunoassay using crude Bt extract-positive sera from asthmatic patients. Evaluation of allergenicity, specific hIgG subclass analysis, and cross- and self-inhibition studies were determined by enzyme-linked immunosorbent assay., Results: Blo t 11 contains multiple IgE and IgG immunodominant peptides scattered throughout the molecule. The dominant IgE and IgG peptides were mapped at amino acid positions 336-557 and 698-875, respectively. An immunodominant peptide (fD) registered a higher percentage of IgE and IgG reactivity compared to the rFL-Blo t 11. Significant serum levels of Blo t 11- and fD-specific IgG1, IgG2 and IgG4, but not IgG3 were detected in the Bt extract-positive sera tested. Cross-inhibition study revealed the rFL-Blo t 11 was significantly inhibited by fD., Conclusion: The IgE and IgG immunodominant peptides of Blo t 11 have been mapped. Our data suggest that utilization of Blo t 11 fragment(s) or chimeric fusion fragments containing IgE and IgG epitopes could be a better alternative in the development of diagnostic and therapeutic reagents for mite allergy.

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.

Background: House dust mite (HDM) Dermatophagoides pteronyssinus is the most important source of indoor allergens that cause allergic diseases in Taiwan. We prepared purified HDM allergens (Der p 1, Der p 2 and Der p 5) to detect allergen-specific immunoglobulin (Ig) E responsiveness among a large number of test subjects. The robust genetic typing system for HLA class II genes also facilitated the study on association of HLA and allergic response toward HDM., Objective: This study intended to investigate the association between HLA class II alleles and the IgE responsiveness to the major allergens from HDM, D. pteronyssinus., Methods: Two hundred and forty-eight subjects were selected for HLA association study. Plasma HDM allergen (Der p 1, Der p 2, Der p 5) -specific IgE and Der p 2-specific IgG antibodies were detected by ELISA, while HLA class II -DRB1, -DQA1, -DQB1, -DPB1 genetic polymorphism was determined by polymerase chain reaction/sequence-specific oligonucleotide probe hybridization (PCR/SSOPH). Statistical comparison of the allelic distribution of each HLA class II genes among the individuals with/without HDM allergen-specific IgE and IgG antibodies were performed., Results: There was no significant association between HLA DRB1, DQB1, DQA1 alleles and HDM-specific IgE responsiveness noted. Only DRB1*0803 and the linked DQA1*0103 alleles showed positive association with Der p 5-specific IgE responsiveness. However, we found that HLA-DPB1*1301 predisposed subjects to IgE responsiveness to HDM Der p 5. HLA DPB1*0501 was weakly associated with the IgE responsiveness to HDM Der p 1 and Der p 5. There was a strong negative association between the HLA-DPB1*0201 allele with IgE responsiveness to Der p 1 (OR: 0.30, P

Group 2 allergens are a major cause of sensitization in patients allergic to house dust mites. This study was performed to determine the prevalence of hypersensitivity to group 2 allergens (Der p 2) of Dermatophagoides pteronyssinus (Dp) in asthmatic patients in Taiwan. To facilitate the analysis of Der p 2-specific IgE, we raised a panel of monoclonal antibodies (MoAbs) to Der p 2 antigens. Purified Der p 2 was obtained after MoAb affinity column purification. There were 82 asthmatic patients (41 adults and 41 children) with hypersensitivity to Dp who were analyzed for hypersensitivity to Der p 2. All of them were both skin test- and serology test-reactive to Dp. Using purified Der p 2, 87.8% (72/82) of patients had a skin-test-positive reaction. Six adults (6/41) and 4 children (4/41) had negative skin tests for Der p 2. Ten families (both parents and children were asthmatics) of the 82 patients were selected for Der p 2 skin testing and Der p 2-specific IgE determination using immunoblot analysis. Results showed that 90% (18/20) of patients' skin reactions to Der p 2 and serum contained specific IgE to Der p 2. Because 87.8% (85.4% of adults and 90.2% of children) of the asthmatic patients with Dp hypersensitivity were allergic to Der p 2, its role in the pathogenesis of asthma in Taiwan appears to be important. Purified Der p 2 allergens can be further used for allergen skin testing and immunotherapy., (Copyright 2000 S. Karger AG, Basel)

Background: A 98-kDa mite paramyosin (Der f 11) from Dermatophagoides farinae (Df) is highly allergenic, and its cDNA (Df642) has been cloned. This paper describes the sequence characteristics and the mapping of the immunodominant human IgE and IgG epitopes of Der f 11., Methods: The protein sequence analysis was performed with a combination of FASTA, GCG, and CLUSTAL W computing packages. The whole cDNA insert and its PCR-derived DNA fragments were generated and expressed in E. coli. These overlapping recombinant peptides (F1 to F5) were used for B-cell epitope mapping with 18 mite-allergic sera by dot immunoassays., Results: Df642 cDNA encodes a partial sequence that contains the 2nd to 26th 28-residue repeats and lacks the N-terminus and the C-terminus. The sequence identity of Der f 11 with other known paramyosins is 34-60%. The dominant IgE epitopes are located in peptides F1 and F4, whereas the dominant IgG epitopes are located in peptides F1 and F2. These peptides are more reactive than whole rDf642., Conclusions: Mite paramyosin is very similar to other known paramyosins. The human IgE and IgG epitopes are scattered throughout the entire molecule. Data also indicate the presence of unique IgE and IgG epitopes in Der f 11.

Blomia tropicalis is an important triggering factor for allergic diseases such as asthma, rhinitis and atopic dermatitis in tropical and subtropical regions, which climate favours the growth of this species. Our previous mite fauna study revealed that Blomia tropicalis is the most dominant species present in Singapore house dust The main objective of this study is to establish a mass culture of Blomia tropicalis for further characterization of the antigenic and molecular properties of this mite. Approximately one gram of mites could be obtained for every 300-gram of culture medium by culturing under natural condition with a mean annual temperature of 30 degrees C and a mean relative humidity of 80%, and harvested by modified Tullgren funnel. Allergen characterization by IgE immunoblot analysis with crude mite extracts showed some IgE reactivity differences between Blomia tropicalis mite extract from Singapore and Colombia. The possible reasons for these findings are the quality and source of the mite protein extracts used, or selective differences in the population under evaluation. Further, the atopic sera tested showed differences in the pattern and Intensity of IgE immunoblot reactivity to crude extracts of Blomia tropicalis and Dermatophagoides pteronyssinus, the other highly prevalent mite in Singapore. These data support the existence of species-specific allergens. In conclusion, we have been successful in setting up B. tropicalis mass cultures and have prepared extracts of high allergenicity.

DNA immunization has been an attractive approach in altering the host immune response to antigen. To examine the utility of DNA immunization in allergic response, we examined the in vivo efficacy of an 'allergen-gene immunization' approach in the modulation of allergen-specific IgE responses in mice. Our results showed first that I.m. injection of a gene construct (pCMVD) containing an important house dust mite allergen gene (Dermatophagoides pteronyssinus group 5 allergen; Der p 5) results in the induction of Der p 5-specific IgG antibodies, but not IgE antibody. We next examined the effect of transduced allergen gene on the expression of specific IgE response in mice after i.p. challenge with recombinant Der p 5 (rDer p 5). Both vector (mock) control- and pCMVD-treated mice were i.p. sensitized with rDer p 5 at 3 weeks after injection of gene construct. Results showed that there is a 90% reduction in the level of specific IgE in pCMVD-treated mice when compared with mock-treated mice. Furthermore, the suppression of specific IgE response can be adoptively transferred with CD8+ T cells from pCMVD-treated mice and such inhibition is in an antigen-specific manner, since the level of specific IgE to an irrelevant allergen, Der p 1, remained unchanged in comparison to that of the mock-treated group. In addition, Der p 5-specific CD8+ T cells could produce high levels of IFN-gamma which probably inhibit allergen-specific IgE responses. Taken together, our results suggest that allergen-gene transfer is effective in the modulation of allergen-specific IgE responses and may provide a novel therapeutic approach.

The efficacy of an "allergen-gene immunization" protocol in altering allergic response was examined. Intramuscular injection of rats with a plasmid DNA encoding a house dust mite allergen into the muscle results in its long-term expression and the induction of specific immune responses. Significantly, this approach prevents the induction of immunoglobulin E synthesis, histamine release in bronchoalveolar fluids, and airway hyperresponsiveness in rats challenged with aerosolized allergen. Furthermore, this suppression is persistent and can be transferred into naive rats by CD8+ T cells from gene-immunized rats. These findings suggest that allergen-gene immunization is effective in modulating allergic responses, and may provide a novel therapeutic approach for allergic diseases.

Background: The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergic patients., Objective: This study was performed to compare the IgE binding activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies., Methods: IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay., Results: A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f7 was used for epitope analysis. All of these reacted with each allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, four could inhibit the binding of IgE (WH9, WH22, WP8 and HD19) while one (WH14) could not., Conclusions: Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specificities.

A lambda gt11 library for cDNA from Dermatophagoides pteronyssinus was screened by plaque immunoassay, and a number of related clones that produced IgE-binding proteins were isolated. Their sequences were homologous to that of a cDNA described previously, which contained a reading frame encoding a 17 kd polypeptide and which as determined by serologic analysis corresponded to a protein with relative molecular mass of 14 kd in mite extracts. The cDNA from the lambda gt11 clones was truncated so it was used to obtain longer clones from a lambda gt10 library. Analysis of the sequence of these clones showed that the allergen now designated Der p V is produced from a 132-residue polypeptide, which has a putative 19-residue leader peptide and a 113-residue mature protein. This would have a molecular weight of 14 kd, corresponding to that found in mite extracts. IgE binding studies with the lambda gt11 clone and a fusion of the mature sequence in a pGEX construct showed that it reacted with 50% of allergic sera. Further studies with skin tests indicated that it caused reactions in about 60% of patients with asthma and 29% of those with allergic rhinitis alone.

The production of allergens from cDNA clones will provide a clonally pure source of material for experimental and perhaps clinical studies. Attempts to produce the major mite allergen, Der p I, in a highly antigenic form in bacteria have, to date, had limited success. In this study, a high level of production of Der p I from a Cup1 gene cassette from pYELC5-13T in Saccharomyces cerevisiae is described. Although the protein was insoluble, it could be readily solubilized in a urea solution and remained in solution when it was returned to more physiologic buffers. An amount equivalent to about 1 mg/L of yeast culture could then be isolated by affinity chromatography with an immobilized monoclonal antibody. This product reacted strongly with IgE in 9/11 sera from mite-allergic patients compared to the 50% reactivity achieved for Der p I previously produced as a fusion by bacteria. Similarly, the intensity of binding and ability to absorb out Der p I specificities were much greater for the yeast, pYELC5-13T, product. Studies with monoclonal antibodies also demonstrated the yeast, Der p I, had a high degree of antigenicity, although clear differences with the native allergen were demonstrated. The high frequency of reactivity with IgE of the pYELC5-13T formally demonstrates that a single gene product of Der p I is a major allergen and demonstrates that even for Der p I, which is synthesized from a proenzyme, considerable antigenicity can be obtained by expressing the mature protein.

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.

Lambda gt11 clones expressing the major house dust mite allergen, Der p II, have been reported to react with IgE in the serum of a high proportion of allergic patients. The clones described, however, only produced small quantities of protein which was not fused to the beta-galactosidase of the vector. A construct of the Der p II is described which produces a fusion of Der p II, minus its leader sequence, with the glutathione-S transferase in the pGEX vector. This could be readily isolated and was shown to react with IgE in 22 of 24 patient sera showing reactivity to native Der p II, the sera not reacting having low reactivity to native protein. Absorption analysis showed that the recombinant material removed most of the IgE reactivity of patients to native Der p II. The construct described, therefore, should be valuable for quantitative studies of a pure mite allergen.

A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.

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. [ABSTRACT FROM AUTHOR]

Analysis by one-dimensional (1-D) SDS-PAGE/Western blotting of whole mite extract (larval and adult stages) with sheep sera taken 0–84 days after infection with the sheep scab mite,Psoroptes ovisrevealed a progressive IgE antibody response, with a dominant high molecular weight allergen (MW 180 kDa) detected early during infection. Further analysis by two-dimensional (2-D) SDS-PAGE/Western blotting with post-infection sera, revealed a more complex picture with numerous (> 20) IgE reactive spots. Trypsin digest and Maldi-ToF analyses of these spots identified two house dust mite allergen homologues, namely tropomyosin (Der p 10) and paramyosin (Der p 11), and analysis of a third spot indicated an apolipoprotein-like IgE reactive protein (Der p 14). Further 1-D and 2-D SDS/Western blotting, with specific antibodies to the house dust mite allergens Der p 10, 11, and to the IgE reactive peptide of the high molecular weight house dust mite allergen, Der p 14 (vitellogenin/apolipophorin), provided firm evidence for the presence of these three allergens in extracts of thePsoroptesmite. These studies show for the first time that homologues of the house dust mite 10, 11 and 14 group allergens are represented as immunodominant allergens of the sheep scab mite, and may represent important vaccine candidates. [ABSTRACT FROM AUTHOR]

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.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The major mite allergen Der p II shows marked resistance to denaturation and is expressed from cDNA in bacteria with almost all of its IgE binding activity. Despite these properties, the IgE binding activity appears to be dependent on maintaining the complete primary structure. Random fragment libraries of cDNA, able to code for up to 93 of the 129 amino acid residue protein, did not express IgE binding peptides. Large overlapping peptides 1-69, 69-129 and 42-117 expressed as the fusions from the glutathione transferase of pGEX vectors only had binding activity with IgE in 15 out of 57 sera, and this was typically weak. Sera from children with atopic dermatitis bound IgE in seven out of eight cases but this was also weak compared with their strong reactivity to intact recombinant Derp II. The inability of such large peptides to form IgE binding structures suggests that the antigenic determinants of Der p II are highly conformational and restricted. [ABSTRACT FROM AUTHOR]