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Biochemical Characterization of Intact Allergen Extracts and Chemically Modified Allergoids Representing Grass, Birch and House Dust Mite Allergen Extracts
S118 Abstracts
Biochemical Characterization of Intact Allergen Extracts and Chemically Modified Allergoids Representing Grass, Birch and House Dust Mite Allergen Extracts H. Henmar, K. Meno, L. Friberg, G. N. Hansen, A. Giselsson, H. Ipsen; Research Department, ALK-Abelló A/S, Hørsholm, DENMARK. RATIONALE: The allergoid concept was developed in the US in the seventies. The rationale was to chemically modify allergens thereby decreasing allergenicity while maintaining immunogenicity. We have investigated the biochemical changes of three different allergen extracts when treated with glutaraldealdehyde. METHODS: Three different extracts were investigated, i.e. Phleum pratense, Betula verrucosa and Dermatophagoides pteronyssinus. The intact and modified extracts were characterized by size exclusion chromatography, crossed immuno-electrophoresis (CIE), SDS-PAGE and immunoblotting. Solid phase IgE inhibition immuno-assays were performed on the ADVIA Centaur platform. RESULTS: The intact allergen extracts showed a variable pattern in size exclusion chromatography, however, after chemical modification all extracts showed the same high molecular weight pattern. Thus, a change in size and also in charge is observed when the proteins are modified using glutaraldehyde.CIE experiments showed that some protein/allergens are affected more than others as Der p 1 seemed to be less affected by the glutaraldehyde treatment than did Der p 2. The hill slopes calculated from the IgE inhibition assays were significantly changed when modifying the allergen extracts. CONCLUSIONS: Chemical treatment of an allergen extract with glutaraldehyde changes the size and charge of the proteins. Major allergens are differently affected by the treatment. Significantly different hill slopes in the IgE inhibition assays indicate a change in epitope composition. Due to changes in epitope composition clinical documentation from immunotherapy with intact allergen extracts can not be used as documentation of modified allergen extracts.
460
SUNDAY
Cloning and Characterization of Art v 2 from Artemisia Vulgaris Pollen J. Asturias1, C. Arilla1, I. Ibarrola1, J. Daza2, Y. Puente3, A. Martinez1; 1R & D, Bial-Arístegui, Bilbao, SPAIN, 2Alergia, Hospital Virgen Macarena, Sevilla, SPAIN, 3Alergoclínica Virgen de Loreto, Córdoba, SPAIN. RATIONALE: Artemisia vulgaris (mugwort) belongs to the same family than ragweed, the Compositae or Asteracea family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. METHODS: Skin prick tests were performed in twelve patients allergic to mugwort and 10 control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Expression of Art v 2 was achieved using vector pKN172 and E. coli BL21 (DE3). RESULTS: Skin prick tests showed Art v 2 prevalences of 58% at 200 g/ml or 33% at 50 g/ml, whereas none false positives were detected among control patients. Purified nArt v 2 had 33 kDa and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search using BLAST software demonstrated that Art v 2 share 55-42% identical residues with pathogenesis related protein PR-1 of tomate, potato, rape, wheat, and rice. Homology was also found to Ves v 5 (41% identical residues). CONCLUSIONS: Art v 2 from mugwort is the first pollen allergen that belongs to the pathogenesis related protein PR-1.
461
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Potential Alternative ELISA Assays to Detect Alt A1 Using IgM and IgG Antibodies M. Abebe, V. Kumar, S. Sevinc, H. M. Vijay; Environmental Health, Health Canada, Ottawa, ON, CANADA. RATIONALE: The association of asthma and allergies with Alternaria alternata is well established. A two-site sandwich ELISA assay is used to detect Alt a1, a doublet protein molecule, in environmental samples. The ELISA method presently used utilizes IgG2a as primary and secondary antibody. In this study we report the possibility of developing ELISA assays that can utilize either IgM or a combination of IgM and IgG to detect the allergen providing alternative techniques to detect Alt a1. METHODS: Two IgM monoclonal antibodies, against recombinant Alt a1 (r-IgM) and native Alt a1 (n-IgM), and a commercially available IgG2a were used, either alone or in combination, to detect Alt a1 in a twosite sandwich ELISA. Atopic IgE was included in the IgM based ELISA tests. RESULTS: The r-IgM based ELISA detected recombinant Alt a1 only while a combination of the r-IgM and IgE detected both the recombinant and native allergen. A combination of r-IgM and IgG2a did not detect the allergen in either state. A combination of n-IgM and IgE based ELISA detected the native allergen, while a combination of r-IgM and n-IgM and IgG2a ELISA tests did not detect the allergen in either state. CONCLUSIONS: The fact that the sandwich ELISA tests based on the r-IgM and IgE as well as the n-IgM and IgE are able to detect the native and the recombinant allergen indicate the possible existence of epitopes against which IgG as well as IgM antibodies can be used to detect Alt a1 in a sandwich ELISA. Funding: Health Canada
462
Epitope Mapping of Der p 2 by Site-directed Mutagenesis: Differential IgE Binding Epitope Profiles among Individuals Sensitized to Only Dermatophagoides spp. and those with Non-pyroglyphid Mite Responses K. Reginald, F. T. Chew; Department of Biological Science, National University of Singapore, Singapore, SINGAPORE. RATIONALE: Der p 2 is a major allergen of the Dermatophagoides pteronyssinus dust mite. Epitope mapping of Der p 2 will allow the design of précised hypoallergenic immunotherapeutic molecules. METHODS: Thirty two different single site-directed alanine mutants were generated, expressed and purified under denaturing conditions, and refolded by rapid dilution into 50mM sodium acetate, pH4.6. Atopic individuals evaluated in this study were identified by a positive skin prick test to rDer p 2. IgE binding capacity of the mutants was evaluated via ELISA, with Der p 2 as the positive control. Additionally, these individuals were also screened for their sensitization to other orthologs of group 2 allergens from 7 other mite species to assess the influence of such sensitization to the epitope profile. RESULTS: Differential IgE binding capacities to the various mutants were observed based on the profile of responses to the different group 2 allergens. Individuals who only responded to Der p 2 and Der f 2 showed significantly reduced IgE binding towards mutant E102A. Individuals who responded to group 2 allergens from both pyroglyphid and nonpyroglyphid mites showed significant IgE binding reduction to mutants H30A, E102A and N114A. In a third group, individuals who had stronger IgE binding to group 2 allergens from non-pyroglyphid mites compared to Der p 2 showed low IgE binding to mutants N114A, E102A, L37A, V104A, V106A and K77A. CONCLUSIONS: Differential IgE binding epitopes on Der p 2 were observed based on the atopic individuals’ sensitization profile to group 2 allergens from the different dust mites. Funding: Biomedical Research Council (BMRC) Singapore
463
Biochemical Characterization of Intact Allergen Extracts and Chemically Modified Allergoids Representing Grass, Birch and House Dust Mite Allergen Extracts H. Henmar, K. Meno, L. Friberg, G. N. Hansen, A. Giselsson, H. Ipsen; Research Department, ALK-Abelló A/S, Hørsholm, DENMARK. RATIONALE: The allergoid concept was developed in the US in the seventies. The rationale was to chemically modify allergens thereby decreasing allergenicity while maintaining immunogenicity. We have investigated the biochemical changes of three different allergen extracts when treated with glutaraldealdehyde. METHODS: Three different extracts were investigated, i.e. Phleum pratense, Betula verrucosa and Dermatophagoides pteronyssinus. The intact and modified extracts were characterized by size exclusion chromatography, crossed immuno-electrophoresis (CIE), SDS-PAGE and immunoblotting. Solid phase IgE inhibition immuno-assays were performed on the ADVIA Centaur platform. RESULTS: The intact allergen extracts showed a variable pattern in size exclusion chromatography, however, after chemical modification all extracts showed the same high molecular weight pattern. Thus, a change in size and also in charge is observed when the proteins are modified using glutaraldehyde.CIE experiments showed that some protein/allergens are affected more than others as Der p 1 seemed to be less affected by the glutaraldehyde treatment than did Der p 2. The hill slopes calculated from the IgE inhibition assays were significantly changed when modifying the allergen extracts. CONCLUSIONS: Chemical treatment of an allergen extract with glutaraldehyde changes the size and charge of the proteins. Major allergens are differently affected by the treatment. Significantly different hill slopes in the IgE inhibition assays indicate a change in epitope composition. Due to changes in epitope composition clinical documentation from immunotherapy with intact allergen extracts can not be used as documentation of modified allergen extracts.
460
SUNDAY
Cloning and Characterization of Art v 2 from Artemisia Vulgaris Pollen J. Asturias1, C. Arilla1, I. Ibarrola1, J. Daza2, Y. Puente3, A. Martinez1; 1R & D, Bial-Arístegui, Bilbao, SPAIN, 2Alergia, Hospital Virgen Macarena, Sevilla, SPAIN, 3Alergoclínica Virgen de Loreto, Córdoba, SPAIN. RATIONALE: Artemisia vulgaris (mugwort) belongs to the same family than ragweed, the Compositae or Asteracea family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. METHODS: Skin prick tests were performed in twelve patients allergic to mugwort and 10 control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Expression of Art v 2 was achieved using vector pKN172 and E. coli BL21 (DE3). RESULTS: Skin prick tests showed Art v 2 prevalences of 58% at 200 g/ml or 33% at 50 g/ml, whereas none false positives were detected among control patients. Purified nArt v 2 had 33 kDa and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search using BLAST software demonstrated that Art v 2 share 55-42% identical residues with pathogenesis related protein PR-1 of tomate, potato, rape, wheat, and rice. Homology was also found to Ves v 5 (41% identical residues). CONCLUSIONS: Art v 2 from mugwort is the first pollen allergen that belongs to the pathogenesis related protein PR-1.
461
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Potential Alternative ELISA Assays to Detect Alt A1 Using IgM and IgG Antibodies M. Abebe, V. Kumar, S. Sevinc, H. M. Vijay; Environmental Health, Health Canada, Ottawa, ON, CANADA. RATIONALE: The association of asthma and allergies with Alternaria alternata is well established. A two-site sandwich ELISA assay is used to detect Alt a1, a doublet protein molecule, in environmental samples. The ELISA method presently used utilizes IgG2a as primary and secondary antibody. In this study we report the possibility of developing ELISA assays that can utilize either IgM or a combination of IgM and IgG to detect the allergen providing alternative techniques to detect Alt a1. METHODS: Two IgM monoclonal antibodies, against recombinant Alt a1 (r-IgM) and native Alt a1 (n-IgM), and a commercially available IgG2a were used, either alone or in combination, to detect Alt a1 in a twosite sandwich ELISA. Atopic IgE was included in the IgM based ELISA tests. RESULTS: The r-IgM based ELISA detected recombinant Alt a1 only while a combination of the r-IgM and IgE detected both the recombinant and native allergen. A combination of r-IgM and IgG2a did not detect the allergen in either state. A combination of n-IgM and IgE based ELISA detected the native allergen, while a combination of r-IgM and n-IgM and IgG2a ELISA tests did not detect the allergen in either state. CONCLUSIONS: The fact that the sandwich ELISA tests based on the r-IgM and IgE as well as the n-IgM and IgE are able to detect the native and the recombinant allergen indicate the possible existence of epitopes against which IgG as well as IgM antibodies can be used to detect Alt a1 in a sandwich ELISA. Funding: Health Canada
462
Epitope Mapping of Der p 2 by Site-directed Mutagenesis: Differential IgE Binding Epitope Profiles among Individuals Sensitized to Only Dermatophagoides spp. and those with Non-pyroglyphid Mite Responses K. Reginald, F. T. Chew; Department of Biological Science, National University of Singapore, Singapore, SINGAPORE. RATIONALE: Der p 2 is a major allergen of the Dermatophagoides pteronyssinus dust mite. Epitope mapping of Der p 2 will allow the design of précised hypoallergenic immunotherapeutic molecules. METHODS: Thirty two different single site-directed alanine mutants were generated, expressed and purified under denaturing conditions, and refolded by rapid dilution into 50mM sodium acetate, pH4.6. Atopic individuals evaluated in this study were identified by a positive skin prick test to rDer p 2. IgE binding capacity of the mutants was evaluated via ELISA, with Der p 2 as the positive control. Additionally, these individuals were also screened for their sensitization to other orthologs of group 2 allergens from 7 other mite species to assess the influence of such sensitization to the epitope profile. RESULTS: Differential IgE binding capacities to the various mutants were observed based on the profile of responses to the different group 2 allergens. Individuals who only responded to Der p 2 and Der f 2 showed significantly reduced IgE binding towards mutant E102A. Individuals who responded to group 2 allergens from both pyroglyphid and nonpyroglyphid mites showed significant IgE binding reduction to mutants H30A, E102A and N114A. In a third group, individuals who had stronger IgE binding to group 2 allergens from non-pyroglyphid mites compared to Der p 2 showed low IgE binding to mutants N114A, E102A, L37A, V104A, V106A and K77A. CONCLUSIONS: Differential IgE binding epitopes on Der p 2 were observed based on the atopic individuals’ sensitization profile to group 2 allergens from the different dust mites. Funding: Biomedical Research Council (BMRC) Singapore
463