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Comparison Of Total Protein Profile Of Alternaria Alternata Extract Obtained From Various U.S. Allergenic Extract Manufacturers
AB100 Abstracts
346
SUNDAY
Comparison Of Total Protein Profile Of Alternaria Alternata Extract Obtained From Various U.S. Allergenic Extract Manufacturers Dr. Jay E. Slater, MD1, Ms. Allison Zoch2, Ms. Shoshana Newman-Gerhardt2, Dr. Taruna Khurana, PhD3; 1FDA/CBER/OVRR/DBPAP, Rockville, MD, 2FDA/CBER, 3CBER FDA. RATIONALE: Alternaria alternata sensitization can cause rhinoconjunctivitis and asthma, occasionally fatal. The allergenic extract used for diagnosis and immunotherapy for the condition is non-standardized. Multiple allergens, strain variability, different growth and extraction conditions impart high variability. The purpose of this study is to assess the variability, and to develop a multiplex antibody-based assay for measuring overall potency of A. alternata extracts. METHODS: We prepared A. alternata extract as follows: 5 g of dried, defatted powder was mixed with PBS containing protease inhibitors overnight at 48C. The extract was centrifuged and filtered. In addition, we analyzed six A. alternata allergen extracts from six manufacturers. The extracts were analyzed using 2D gel electrophoresis and GelFree fractionation system. Separated proteins were transferred onto PVDF for human IgE immunoblotting. Proteins detected by IgE were excised from Coomassie stained gels and identified using peptide mass fingerprinting. RESULTS: As expected, A. alternaria allergen extracts exhibited extensive compositional differences by SDS-PAGE and 2D fractionation, and a broad range of Alt a 1 levels (0.1-9.0 mcg/mL) (Alt a 1 ELISA kit, Indoor Biotechnologies). On IgE immunoblot of 2D gel electrophoresis, >10 protein spots were detected, including heat shock protein 70/Alt a 3 (356578610), TBP-associated factor 15B (261336148), beta-glucosidase 2 precursor (380007310) and exoglucanase (6179889). CONCLUSIONS: There are significant composition differences at the protein level in different A. alternata extracts. The range of Al t a 1 levels is broad. We have identified novel allergens that need to be further characterized for the development of a multiplex assay.
347
IgE Antibodies To Cat and Cat Components In Relation To Asthma In a Population Study Of 963 18 Year Olds From Six Schools In Northern Sweden Ms. Hayley James, BS1, Prof. Matthew S. Perzanowski, PhD2, Eva Ronmark, PhD3, Bo Lundback, MD, PhD3, Jillian Roper1, Thomas A. E. Platts-Mills, MD, PhD, FAAAAI4; 1University of Virginia, Charlottesville, VA, 2Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 3Karolinska Institutet, Stockholm, Sweden, 4Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: In Norbotten, the dominant indoor allergens are cat and dog and there is no significant exposure or sensitization to mite, cockroach, Alternaria or Aspergillus. METHODS: Sera were assessed by ImmunoCAP for IgE to cat, dog and horse (dander); birch and timothy pollen; mite, cockroach, Alternaria and Aspergillus. The results were compared to asthma, prevalence and severity. RESULTS: Significant associations with asthma were observed for IgE to animal dander and pollens; only danders remained significant in multivariate _17.5 analysis: odds ratio 5.7 (3.5-9.4) p<0.001, for the association of IgE ab > IU/ml (class 4) and physician diagnosis of asthma. Sera positive for dander were assayed for IgE to Feld1, Feld2 (albumin), Feld4 (lipocalin), and Feld5 w (Cat IgA, alpha-gal): Positive results were seen in 197, 25, 81 and 1 sera respectively. The quantitative results for Feld1, Feld2 and Feld4 correlated with asthma; only Feld1 and Feld4 were significant in multivariate analysis: Odds Ratio 3.0 (1.5 – 5.9) p <0.01, and 4.8 (1.0 – 23) p <0.05 respectively. Interestingly, while Feld1 results correlated most strongly with IgE to cat Rs 0.86 (p <0.001), the results for Feld2 correlated equally, with cat, dog and horse; 0.30, 0.33, 0.26. The results for Feld4 correlated strongly with IgE to horse Rs 0.78, p <0.001, which may reflect the dominance of the lipocalin Equc1 in the horse ImmunoCAP extracts. CONCLUSIONS: The results illustrate the complexity of component analysis for cat and strongly support the importance of both specificity and titer of IgE antibodies.
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
348
Epitope Mapping Of An Anti-Bla g 1 ScFv Used For Cockroach Allergen Quantitation Dr. Geoffrey Mueller, PhD1, Mr. John Ankney2, Dr. Lars Pedersen1, Dr. Taruna Khurana, PhD3, Dr. Jay E. Slater, MD4, Ms. Jill Glesner5, Dr. Anna Pomes, PhD, FAAAAI5, Dr. Robert London1; 1National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 2National Intstitute of Environmental Health Sciences, NIH, 3 FDA Center for Biologics Evaluation and Research, 4FDA/CBER/ OVRR/DBPAP, Rockville, MD, 5Indoor Biotechnologies, Inc., Charlottesville, VA. RATIONALE: Bla g 1 is one of two primary allergens used to measure cockroach allergen exposure. A panel of avian scFv antibodies was developed for composition profiling of whole body cockroach extract. Herein, we mapped the epitope of an anti-Bla g 1 scFv in order to better understand the binding efficiency and cross reactivity with other group 1 cockroach allergens. METHODS: X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was assessed by alanine scanning site-directed mutagenesis and ELISA. The allergen-scFv complex was modeled based on the results. The scFv was tested by ELISA for the ability to block the binding of IgE antibodies from cockroach allergic patients to Bla g 1. RESULTS: Twenty-four rBla g 1-GST alanine mutants were assessed for variations in binding to the scFv compared to wild type. Five mutants showed a significant difference in affinity. These mutations clustered to form a discontinuous epitope comprising four helices of Bla g 1 with high sequence identity to Per a 1. The scFv did not inhibit the interaction of patient IgE antibodies with Bla g 1. CONCLUSIONS: The anti-Bla g 1 scFv is expected to have good detection and quantitation properties for both German and American cockroach species. As the scFv does not interfere with IgE antibody binding, it should act as a good capture antibody for quantifying anti-Bla g 1 serum IgE levels by ELISA.
349
Antigenic Analysis Of The Major Cockroach Allergen Bla g 5 and Its Dust Mite Homolog Der p 8 Ms. Jill Glesner1, Dr. Geoffrey Mueller, PhD2, Dr. Lars Pedersen2, Dr. Martin D. Chapman, PhD, FAAAAI1, Dr. Anna Pomes, PhD, FAAAAI1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC. RATIONALE: Bla g 5 induces the highest IgE antibody (ab) titers compared to cockroach allergens from groups 1, 2, 4 and 7. Bla g 5 and dust mite Der p 8 are glutathione-S-transferases (30% and 26% identical in sequence and surface, respectively). Despite reported IgE ab crossreactivity for GSTs from mite and cockroach, cross-reactivity between both allergens is unknown. METHODS: Bla g 5 and Der p 8 were expressed in Escherichia coli and purified by GST affinity chromatography. Six monoclonal antibodies (mAbs) were raised against Bla g 5. Specific IgE to Der p 1, Der p 2 and Bla g 5 in sera/plasma were measured by ImmunoCAP. Ab binding assays were performed by ELISA. RESULTS: Six anti-Bla g 5 mAbs showed lack of reactivity to Der p 8, and inhibited IgE ab binding to Bla g 5 up to 78%, indicating overlapping epitopes with IgE ab. Fifty two percent (n512/23) of plasma/sera from dust mite allergic patients, with IgE specific for Dermatophagoides pteronyssinus allergens, reacted with Der p 8, and none reacted with Bla g 5. Conversely, 15 sera with Bla g 5 specific IgE did not show significant IgE reactivity to Der p 8. No significant IgE or mAb cross-reactivity was observed between both allergens, in agreement with a low molecular surface homology. CONCLUSIONS: Patients’ IgE binding to Bla g 5 and Der p 8 result from co-sensitization. Immunological analysis of homologous allergens allows evaluation of antigenic cross-reactivity and to assess the relevance of homologous allergens from different sources.
346
SUNDAY
Comparison Of Total Protein Profile Of Alternaria Alternata Extract Obtained From Various U.S. Allergenic Extract Manufacturers Dr. Jay E. Slater, MD1, Ms. Allison Zoch2, Ms. Shoshana Newman-Gerhardt2, Dr. Taruna Khurana, PhD3; 1FDA/CBER/OVRR/DBPAP, Rockville, MD, 2FDA/CBER, 3CBER FDA. RATIONALE: Alternaria alternata sensitization can cause rhinoconjunctivitis and asthma, occasionally fatal. The allergenic extract used for diagnosis and immunotherapy for the condition is non-standardized. Multiple allergens, strain variability, different growth and extraction conditions impart high variability. The purpose of this study is to assess the variability, and to develop a multiplex antibody-based assay for measuring overall potency of A. alternata extracts. METHODS: We prepared A. alternata extract as follows: 5 g of dried, defatted powder was mixed with PBS containing protease inhibitors overnight at 48C. The extract was centrifuged and filtered. In addition, we analyzed six A. alternata allergen extracts from six manufacturers. The extracts were analyzed using 2D gel electrophoresis and GelFree fractionation system. Separated proteins were transferred onto PVDF for human IgE immunoblotting. Proteins detected by IgE were excised from Coomassie stained gels and identified using peptide mass fingerprinting. RESULTS: As expected, A. alternaria allergen extracts exhibited extensive compositional differences by SDS-PAGE and 2D fractionation, and a broad range of Alt a 1 levels (0.1-9.0 mcg/mL) (Alt a 1 ELISA kit, Indoor Biotechnologies). On IgE immunoblot of 2D gel electrophoresis, >10 protein spots were detected, including heat shock protein 70/Alt a 3 (356578610), TBP-associated factor 15B (261336148), beta-glucosidase 2 precursor (380007310) and exoglucanase (6179889). CONCLUSIONS: There are significant composition differences at the protein level in different A. alternata extracts. The range of Al t a 1 levels is broad. We have identified novel allergens that need to be further characterized for the development of a multiplex assay.
347
IgE Antibodies To Cat and Cat Components In Relation To Asthma In a Population Study Of 963 18 Year Olds From Six Schools In Northern Sweden Ms. Hayley James, BS1, Prof. Matthew S. Perzanowski, PhD2, Eva Ronmark, PhD3, Bo Lundback, MD, PhD3, Jillian Roper1, Thomas A. E. Platts-Mills, MD, PhD, FAAAAI4; 1University of Virginia, Charlottesville, VA, 2Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University, New York, NY, 3Karolinska Institutet, Stockholm, Sweden, 4Division of Asthma, Allergy & Immunology, University of Virginia Health System, Charlottesville, VA. RATIONALE: In Norbotten, the dominant indoor allergens are cat and dog and there is no significant exposure or sensitization to mite, cockroach, Alternaria or Aspergillus. METHODS: Sera were assessed by ImmunoCAP for IgE to cat, dog and horse (dander); birch and timothy pollen; mite, cockroach, Alternaria and Aspergillus. The results were compared to asthma, prevalence and severity. RESULTS: Significant associations with asthma were observed for IgE to animal dander and pollens; only danders remained significant in multivariate _17.5 analysis: odds ratio 5.7 (3.5-9.4) p<0.001, for the association of IgE ab > IU/ml (class 4) and physician diagnosis of asthma. Sera positive for dander were assayed for IgE to Feld1, Feld2 (albumin), Feld4 (lipocalin), and Feld5 w (Cat IgA, alpha-gal): Positive results were seen in 197, 25, 81 and 1 sera respectively. The quantitative results for Feld1, Feld2 and Feld4 correlated with asthma; only Feld1 and Feld4 were significant in multivariate analysis: Odds Ratio 3.0 (1.5 – 5.9) p <0.01, and 4.8 (1.0 – 23) p <0.05 respectively. Interestingly, while Feld1 results correlated most strongly with IgE to cat Rs 0.86 (p <0.001), the results for Feld2 correlated equally, with cat, dog and horse; 0.30, 0.33, 0.26. The results for Feld4 correlated strongly with IgE to horse Rs 0.78, p <0.001, which may reflect the dominance of the lipocalin Equc1 in the horse ImmunoCAP extracts. CONCLUSIONS: The results illustrate the complexity of component analysis for cat and strongly support the importance of both specificity and titer of IgE antibodies.
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
348
Epitope Mapping Of An Anti-Bla g 1 ScFv Used For Cockroach Allergen Quantitation Dr. Geoffrey Mueller, PhD1, Mr. John Ankney2, Dr. Lars Pedersen1, Dr. Taruna Khurana, PhD3, Dr. Jay E. Slater, MD4, Ms. Jill Glesner5, Dr. Anna Pomes, PhD, FAAAAI5, Dr. Robert London1; 1National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 2National Intstitute of Environmental Health Sciences, NIH, 3 FDA Center for Biologics Evaluation and Research, 4FDA/CBER/ OVRR/DBPAP, Rockville, MD, 5Indoor Biotechnologies, Inc., Charlottesville, VA. RATIONALE: Bla g 1 is one of two primary allergens used to measure cockroach allergen exposure. A panel of avian scFv antibodies was developed for composition profiling of whole body cockroach extract. Herein, we mapped the epitope of an anti-Bla g 1 scFv in order to better understand the binding efficiency and cross reactivity with other group 1 cockroach allergens. METHODS: X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was assessed by alanine scanning site-directed mutagenesis and ELISA. The allergen-scFv complex was modeled based on the results. The scFv was tested by ELISA for the ability to block the binding of IgE antibodies from cockroach allergic patients to Bla g 1. RESULTS: Twenty-four rBla g 1-GST alanine mutants were assessed for variations in binding to the scFv compared to wild type. Five mutants showed a significant difference in affinity. These mutations clustered to form a discontinuous epitope comprising four helices of Bla g 1 with high sequence identity to Per a 1. The scFv did not inhibit the interaction of patient IgE antibodies with Bla g 1. CONCLUSIONS: The anti-Bla g 1 scFv is expected to have good detection and quantitation properties for both German and American cockroach species. As the scFv does not interfere with IgE antibody binding, it should act as a good capture antibody for quantifying anti-Bla g 1 serum IgE levels by ELISA.
349
Antigenic Analysis Of The Major Cockroach Allergen Bla g 5 and Its Dust Mite Homolog Der p 8 Ms. Jill Glesner1, Dr. Geoffrey Mueller, PhD2, Dr. Lars Pedersen2, Dr. Martin D. Chapman, PhD, FAAAAI1, Dr. Anna Pomes, PhD, FAAAAI1; 1Indoor Biotechnologies, Inc., Charlottesville, VA, 2National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC. RATIONALE: Bla g 5 induces the highest IgE antibody (ab) titers compared to cockroach allergens from groups 1, 2, 4 and 7. Bla g 5 and dust mite Der p 8 are glutathione-S-transferases (30% and 26% identical in sequence and surface, respectively). Despite reported IgE ab crossreactivity for GSTs from mite and cockroach, cross-reactivity between both allergens is unknown. METHODS: Bla g 5 and Der p 8 were expressed in Escherichia coli and purified by GST affinity chromatography. Six monoclonal antibodies (mAbs) were raised against Bla g 5. Specific IgE to Der p 1, Der p 2 and Bla g 5 in sera/plasma were measured by ImmunoCAP. Ab binding assays were performed by ELISA. RESULTS: Six anti-Bla g 5 mAbs showed lack of reactivity to Der p 8, and inhibited IgE ab binding to Bla g 5 up to 78%, indicating overlapping epitopes with IgE ab. Fifty two percent (n512/23) of plasma/sera from dust mite allergic patients, with IgE specific for Dermatophagoides pteronyssinus allergens, reacted with Der p 8, and none reacted with Bla g 5. Conversely, 15 sera with Bla g 5 specific IgE did not show significant IgE reactivity to Der p 8. No significant IgE or mAb cross-reactivity was observed between both allergens, in agreement with a low molecular surface homology. CONCLUSIONS: Patients’ IgE binding to Bla g 5 and Der p 8 result from co-sensitization. Immunological analysis of homologous allergens allows evaluation of antigenic cross-reactivity and to assess the relevance of homologous allergens from different sources.