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Standardization of Allergen Exposure Assessment using Multiplex Fluorescent Array Technology
S178 Abstracts
681
Group 1 Mite Allergen from Dermatophagoides siboney (Der s 1) Behave as Der p 1 and Der f 1 to Detect Mite Allergic Subjects in a D. siboney not Exposed Population E. Scala1, R. Ferrara1, A. Ma´s2, A. Hidalgo2, A. Zaffiro2, D. Pomponi1, A. Labrada2, A. Mari3; 1Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, ITALY, 2Allergen Department–Centro Nacional de Biopreparados, La Habana, CUBA, 3Allergen Department–Centro Nacional de Biopreparados, La Habana, BENIN. RATIONALE: Presence of Dermatophagoides siboney (Der s) in the indoor environment has never been reported in European countries, whereas it has been described as an allergenic source in indoor environments in Cuba. We sought to define whether IgE co-recognition of mite homologous group 1 allergens is present in a non exposed Italian mite allergic population. METHODS: A Der s extract has been used for IgE detection on a singleplex system along with Der p and Der f extracts. Highly purified natural Der s 1, Der f 1, and Der p 1 spotted on a proteomic microarray (ISAC, VBC-Genomics, Austria) have been used for multiplexed IgE detection in 562 subjects having IgE to least one mite allergen using the microarray system. RESULTS: A selected group of mite allergic subjects had IgE detected for Der s extract correlating with IgE values obtained with the Der f and Der p extracts. 54.6, 48.7, and 45.7% of the ISAC tested patients had IgE to Der p 1, Der f 1, and Der s 1, respectively. Correlation index of IgE values was r 5 0.92 and r 5 0.91 when comparing Der s 1 with Der f 1 and Der p 1, respectively, with a p value <0.0001. When stratified by age and gender, percentages did not differ in a statistically significant way. CONCLUSIONS: Sensitization to homologous mite allergens without previous exposure leads to a potential risk of having symptoms while travelling in countries where closely related mite species can be present and release homologous molecules.
682
MONDAY
The Importance of Antibody Cross-Reactivity for the Development of Environmental Immunoassays for Alternaria Alternata D. Schmechel, B. J. Green, F. M. Blachere, E. Janotka, D. H. Beezhold; CDC/Natl. Inst. Occup. Safety & Health, Morgantown, WV. RATIONALE: Alternaria alternata is one of the most common fungi indoors and outdoors and exposure to the fungus has been linked to the severity of asthma and the pathophysiology of chronic rhinosinusitis. A recent national survey of house dust samples concluded that 95-99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies (pAbs) can lack assay specificity and in this study we investigated the cross-reactivity of the pAbs to other common fungal contaminants. METHODS: Reactivity to 24 fungal species typically found in indoor environments was analyzed by inhibition ELISA. The pAbs were also tested by immunoblotting and Halogen immunoassay (HIA) for a subgroup of fungi. RESULTS: Spores of 6 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera and Exserohilum were found to react with the pAbs more strongly than A. alternata when tested by ELISA. Six other fungi reacted in the ELISA at a lower level and 11 fungal species including several Penicillium, Aspergillus, Fusarium and Cladosporium species were not found to inhibit pAb binding. The immunoblots and the HIA staining confirmed that the pAbs react with numerous fungi commonly found in US homes. CONCLUSIONS: Due to extensive cross-reactivity, the ELISA prevalence data previously reported for A. alternata should be considered to represent fungal-specific antigens rather than be interpreted as exclusive contamination with A. alternata alone. The characterization of antibody specificity before application is critical to avoid analytical bias and ambiguities in data interpretation. Funding: CDC/NIOSH
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
683
Characterization of Aerosolized Fel d 1 in a Clinical Model During a One Hour Cat Allergen Challenge (CAC) S. McCue1, L. Nimraj1, V. Walsh1, H. Nandkeshore1, D. Botezayas1, M. Chapman2, J. Corren3, J. Freihaut4, P. Patel1, A. Salapatek1; 1Allied Research International-Cetero Research, Mississauga, ON, CANADA, 2 Indoor Biotechnologies, Charlottesville, VA, 3Allergy Research Foundation, Los Angeles, CA, 4Pennsylvania State University, Philadelphia, PA. RATIONALE: We have developed a natural cat allergen challenge model where allergic/asthmatic subjects will be exposed to consistent levels of transient inhalable Fel d 1 (500-1200 6 300 ng/m3) for one hour. METHODS: Fel d 1, extracted from milled cat pelts was initially aerosolized and then maintained by two in-living cats. The CACR is carpeted, contains a subject chair and a couch covered with a cotton sheet used to transiently release allergen. Allergen was measured using personal air samplers in 15 minute increments for one hour and analyzed using ELISA. RESULTS: Three hours of Fel d 1 aerosolization and 9 weeks of in-living cats in the CAC room resulted in a mean transient inhalable level of Fel d 1 of 903 6 574 ng/m3. During a 1-hour challenge, the mean concentration of inhalable (0-25mm) Fel d 1 was 1406 6 460 ng/m3, 0-15 minutes: 4205 6 2068 ng/m3, 15-30 minutes: 735 6 361 ng/m3, 30-45 minutes: 439 6 44 ng/m3, 45-60 minutes: 243 6 88 ng/m3. The mean concentration of thoracic Fel d 1(0-10mm) in 60 minutes: 1365 6 115 ng/m3, 0-15 minutes 4040 6 410 ng/m3, 15-30 minutes: 772 6 70 ng/m3, 30-45 minutes: 432 6 32 ng/m3, 45-60 minutes: 215 6 12 ng/m3. The concentration of respirable Fel d 1 (0-2.5mm) in 60 minutes: 267 ng/m3, 0-15 minutes 1745 6 183 ng/m3, 15-30 minutes: 781 6 45 ng/m3, 30-45 minutes: 75 6 75 ng/m3, 45-60 minutes: 19 6 19 ng/m3. CONCLUSIONS: In this transient exposure model, subject exposure to allergen is greatest initially with lower mean exposure after the hour and is representative of exposures observed naturally. Funding: Allied Research International
684
Standardization of Allergen Exposure Assessment using Multiplex Fluorescent Array Technology E. M. King1, S. Filep1, C. Earle1, C. Bronzert2, R. Hamilton2, M. D. Chapman1; 1INDOOR Biotechnologies Inc., Charlottesville, VA, 2DACI Laboratory, Johns Hopkins University, Baltimore, MD. RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. Here, we have compared results obtained by the same operator as well as between four different operators to evaluate reproducibility of a fluorescent multiplex array for eight allergens. METHODS: Intra-operator reproducibility of an 8-plex fluorescent array measuring Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1 and Bla g 2 was determined by measuring a total of 15 house dust and air filter extracts by the same operator on two different days. Reproducibility of 8-plex results between operators was determined by measurement of the same 15 samples by 4 different operators. RESULTS: The results for all analyses and operators agreed in their assessment of samples as being below or above the limit of detection. Results obtained by the same operator on different occasions were reproducible for all analytes with coefficients of variance (CVs) ranging between 2.5% and 7.3% (mean CV 5 4.4%). Inter-operator reproducibility for all analytes was also good with CVs ranging from 10.3% to 20.6% (mean CV 5 13.3%). CONCLUSIONS: The data indicate that the fluorescent multiplex array produces results that are reproducible within and between operators and which will improve standardization of allergen exposure assessment. Funding: NIEHS SBIR contract ES 55545
681
Group 1 Mite Allergen from Dermatophagoides siboney (Der s 1) Behave as Der p 1 and Der f 1 to Detect Mite Allergic Subjects in a D. siboney not Exposed Population E. Scala1, R. Ferrara1, A. Ma´s2, A. Hidalgo2, A. Zaffiro2, D. Pomponi1, A. Labrada2, A. Mari3; 1Center for Clinical and Experimental Allergology, IDI-IRCCS, Rome, ITALY, 2Allergen Department–Centro Nacional de Biopreparados, La Habana, CUBA, 3Allergen Department–Centro Nacional de Biopreparados, La Habana, BENIN. RATIONALE: Presence of Dermatophagoides siboney (Der s) in the indoor environment has never been reported in European countries, whereas it has been described as an allergenic source in indoor environments in Cuba. We sought to define whether IgE co-recognition of mite homologous group 1 allergens is present in a non exposed Italian mite allergic population. METHODS: A Der s extract has been used for IgE detection on a singleplex system along with Der p and Der f extracts. Highly purified natural Der s 1, Der f 1, and Der p 1 spotted on a proteomic microarray (ISAC, VBC-Genomics, Austria) have been used for multiplexed IgE detection in 562 subjects having IgE to least one mite allergen using the microarray system. RESULTS: A selected group of mite allergic subjects had IgE detected for Der s extract correlating with IgE values obtained with the Der f and Der p extracts. 54.6, 48.7, and 45.7% of the ISAC tested patients had IgE to Der p 1, Der f 1, and Der s 1, respectively. Correlation index of IgE values was r 5 0.92 and r 5 0.91 when comparing Der s 1 with Der f 1 and Der p 1, respectively, with a p value <0.0001. When stratified by age and gender, percentages did not differ in a statistically significant way. CONCLUSIONS: Sensitization to homologous mite allergens without previous exposure leads to a potential risk of having symptoms while travelling in countries where closely related mite species can be present and release homologous molecules.
682
MONDAY
The Importance of Antibody Cross-Reactivity for the Development of Environmental Immunoassays for Alternaria Alternata D. Schmechel, B. J. Green, F. M. Blachere, E. Janotka, D. H. Beezhold; CDC/Natl. Inst. Occup. Safety & Health, Morgantown, WV. RATIONALE: Alternaria alternata is one of the most common fungi indoors and outdoors and exposure to the fungus has been linked to the severity of asthma and the pathophysiology of chronic rhinosinusitis. A recent national survey of house dust samples concluded that 95-99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Polyclonal antibodies (pAbs) can lack assay specificity and in this study we investigated the cross-reactivity of the pAbs to other common fungal contaminants. METHODS: Reactivity to 24 fungal species typically found in indoor environments was analyzed by inhibition ELISA. The pAbs were also tested by immunoblotting and Halogen immunoassay (HIA) for a subgroup of fungi. RESULTS: Spores of 6 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera and Exserohilum were found to react with the pAbs more strongly than A. alternata when tested by ELISA. Six other fungi reacted in the ELISA at a lower level and 11 fungal species including several Penicillium, Aspergillus, Fusarium and Cladosporium species were not found to inhibit pAb binding. The immunoblots and the HIA staining confirmed that the pAbs react with numerous fungi commonly found in US homes. CONCLUSIONS: Due to extensive cross-reactivity, the ELISA prevalence data previously reported for A. alternata should be considered to represent fungal-specific antigens rather than be interpreted as exclusive contamination with A. alternata alone. The characterization of antibody specificity before application is critical to avoid analytical bias and ambiguities in data interpretation. Funding: CDC/NIOSH
J ALLERGY CLIN IMMUNOL FEBRUARY 2008
683
Characterization of Aerosolized Fel d 1 in a Clinical Model During a One Hour Cat Allergen Challenge (CAC) S. McCue1, L. Nimraj1, V. Walsh1, H. Nandkeshore1, D. Botezayas1, M. Chapman2, J. Corren3, J. Freihaut4, P. Patel1, A. Salapatek1; 1Allied Research International-Cetero Research, Mississauga, ON, CANADA, 2 Indoor Biotechnologies, Charlottesville, VA, 3Allergy Research Foundation, Los Angeles, CA, 4Pennsylvania State University, Philadelphia, PA. RATIONALE: We have developed a natural cat allergen challenge model where allergic/asthmatic subjects will be exposed to consistent levels of transient inhalable Fel d 1 (500-1200 6 300 ng/m3) for one hour. METHODS: Fel d 1, extracted from milled cat pelts was initially aerosolized and then maintained by two in-living cats. The CACR is carpeted, contains a subject chair and a couch covered with a cotton sheet used to transiently release allergen. Allergen was measured using personal air samplers in 15 minute increments for one hour and analyzed using ELISA. RESULTS: Three hours of Fel d 1 aerosolization and 9 weeks of in-living cats in the CAC room resulted in a mean transient inhalable level of Fel d 1 of 903 6 574 ng/m3. During a 1-hour challenge, the mean concentration of inhalable (0-25mm) Fel d 1 was 1406 6 460 ng/m3, 0-15 minutes: 4205 6 2068 ng/m3, 15-30 minutes: 735 6 361 ng/m3, 30-45 minutes: 439 6 44 ng/m3, 45-60 minutes: 243 6 88 ng/m3. The mean concentration of thoracic Fel d 1(0-10mm) in 60 minutes: 1365 6 115 ng/m3, 0-15 minutes 4040 6 410 ng/m3, 15-30 minutes: 772 6 70 ng/m3, 30-45 minutes: 432 6 32 ng/m3, 45-60 minutes: 215 6 12 ng/m3. The concentration of respirable Fel d 1 (0-2.5mm) in 60 minutes: 267 ng/m3, 0-15 minutes 1745 6 183 ng/m3, 15-30 minutes: 781 6 45 ng/m3, 30-45 minutes: 75 6 75 ng/m3, 45-60 minutes: 19 6 19 ng/m3. CONCLUSIONS: In this transient exposure model, subject exposure to allergen is greatest initially with lower mean exposure after the hour and is representative of exposures observed naturally. Funding: Allied Research International
684
Standardization of Allergen Exposure Assessment using Multiplex Fluorescent Array Technology E. M. King1, S. Filep1, C. Earle1, C. Bronzert2, R. Hamilton2, M. D. Chapman1; 1INDOOR Biotechnologies Inc., Charlottesville, VA, 2DACI Laboratory, Johns Hopkins University, Baltimore, MD. RATIONALE: Standardization of allergen exposure measurements is important to ensure reproducibility of results obtained by different laboratories. Here, we have compared results obtained by the same operator as well as between four different operators to evaluate reproducibility of a fluorescent multiplex array for eight allergens. METHODS: Intra-operator reproducibility of an 8-plex fluorescent array measuring Der p 1, Der f 1, Mite Group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1 and Bla g 2 was determined by measuring a total of 15 house dust and air filter extracts by the same operator on two different days. Reproducibility of 8-plex results between operators was determined by measurement of the same 15 samples by 4 different operators. RESULTS: The results for all analyses and operators agreed in their assessment of samples as being below or above the limit of detection. Results obtained by the same operator on different occasions were reproducible for all analytes with coefficients of variance (CVs) ranging between 2.5% and 7.3% (mean CV 5 4.4%). Inter-operator reproducibility for all analytes was also good with CVs ranging from 10.3% to 20.6% (mean CV 5 13.3%). CONCLUSIONS: The data indicate that the fluorescent multiplex array produces results that are reproducible within and between operators and which will improve standardization of allergen exposure assessment. Funding: NIEHS SBIR contract ES 55545