Dust mite infestation of flour samples.

BACKGROUND: Ingestion of flour contaminated with dust mite may trigger severe anaphylaxis in tropical and sub-tropical regions. AIMS: This study aimed to evaluate environmental factors that affect dust mite propagation in the tropics. MATERIALS & METHODS: Dust mites were introduced to a variety of flour samples and incubated at two different environmental conditions. RESULTS: It was found that dust mites populations flourished best in wheat flour compared to other varieties of flour, and at ambient temperatures with high humidity instead of the air conditioned environment. CONCLUSION: Dust mite infestation of flour is dependent on the presence of wheat and high ambient temperature in the tropics.

Anaphylaxis following the ingestion of flour contaminated by house dust mites--a report of two cases from Singapore.

This study presents two patients who developed anaphylaxis after eating mite-contaminated food, and also contains a survey of dust-mites contamination in flour samples from Singapore households. The clinical records of each patient was studied. Patient A developed anaphylaxis twenty minutes following the ingestion of home-made fried fish coated with Japanese flour, while Patient B developed similar life-threatening symptoms one hour after the ingestion of home baked scones. Both patients were NSAID-intolerant and had a history of allergic rhinitis. Skin prick tests showed a strong positive result for dust-mites and for extracts prepared from the ingested flour. Flour samples were also examined microscopically which revealed large numbers of live Dermatophagoides farinae dust-mites. A survey of 57 flour samples showed that 4 samples (7%) were contaminated with dust mites. The findings in the present study confirm that mite-contamination of flour exists in Singaporean households, and it may trigger anaphylaxis in susceptible individuals.

Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.

BACKGROUND: Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples. RESULTS: A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust. CONCLUSIONS: A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.

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.

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.

Immunochemical characterization of edible bird's nest allergens.

BACKGROUND: We have previously described anaphylaxis induced by edible bird's nest (BN) and demonstrated that this condition is IgE mediated. OBJECTIVES: This study aimed at describing the immunochemical properties of the BN allergens. Comparative studies between 3 commercially available sources (according to the country of origin) of BN were also made. METHODS: Crude extracts of commercially available processed BN from Sarawak (Malaysia), Thailand, and Indonesia and fresh unprocessed BN from the caves of Sarawak were obtained by means of aqueous extraction. Specific IgE toward these sources were determined by using fluorescence allergosorbent tests (FASTs). Cross-reactivity studies between the 3 sources of commercially available processed BN were carried out by means of FAST inhibition. Immunochemical characterization by means of IgE immunoblot, periodate treatment, and heat stability studies were carried out on fresh unprocessed BN from Sarawak. RESULTS: Serum from allergic patients showed differences in IgE binding to the 3 sources of commercially available BN, with the highest levels of specific IgE recorded with the Sarawak source (P <.0001). Of these, only the Sarawak and Thailand sources showed considerable cross-reactivity. Further work on the unprocessed fresh Sarawak source identified a putative 66-kd major allergen containing several isoforms. Periodate treatment resulted in loss of IgE binding. Despite a progressive decline in the molecular weights of allergens on SDS-PAGE with increasing periods of boiling, IgE binding, as assessed by means of FAST, was not affected. N-terminal sequence of the major putative allergen (66 kd) showed homology to a domain of an ovoinhibitor precursor in chicken (SWISS-PROT accession No. P10184). CONCLUSIONS: We have described the immunochemical properties of BN allergens. Edible BN from different sources are allergenically dissimilar. The putative major allergen is a 66-kd protein.

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.

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.

Culture of Blomia tropicalis and IgE immunoblot characterization of its allergenicity.

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.

Prevalence and distribution of indoor allergens in Singapore.

BACKGROUND AND AIMS: Immediate hypersensitivity to indoor allergens is known to be associated with allergic asthma. This study evaluated the prevalence and distribution of six indoor allergens in 956 dust samples obtained from homes, childcare centres, schools, and a hospital in tropical Singapore. Seasonality of mite allergens was also assessed. METHODS: The major allergens of the Dermatophagoides spp. dust mites, Der p 1 and Der f 1; major cat and dog allergens, Can f 1 (dog) and Fel d 1 (cat); and cockroach, Bla g 1, were measured by specific enzyme immunoassays. Allergen levels of the storage mite, Blomia tropicalis (Blo t), were measured by a fluorescent allergosorbent test (FAST) inhibition assay. RESULTS: Our results showed that homes had significantly higher concentrations and prevalence of allergens compared with the other locations, except for Bla g 1, where higher mean levels were found in schools. Within the homes, the highest concentrations of mite allergens were found in mattresses (geometric mean: 1.2 microg/g dust Der p 1; 2717 Allergen Units per gram dust [AU/g] Blo t), and carpets (1.5 microg/g Der p 1; 1620 AU/g Blo t), whilst Bla g 1 was mainly concentrated in the storerooms (geometric mean = 3.5 units/g) and kitchens (geometric mean = 5.1 units/g). The major cat and dog allergens were well distributed and not confined to homes with pets. Their highest levels were found in dust of soft furnishings, carpets and mattresses. There was an absence of significant seasonal variation in Der p 1, Der f 1 and Blo t levels in the homes over a 1 year period. CONCLUSION: The results indicate that compared with public places, the home consitutes a major reservior of indoor allergens. Allergens of the storage mite, B. tropicalis, should be considered as a major allergenic component of dust in Singapore.