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Screening for Conformational Epitopes Using Heat Denaturation of Allergens on ELISA Plates
Abstracts S117
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
Use of Cross-reacting Carbohydrate Determinants (CCD) in Specific IgE Detection U. BANIK; Product Support Allergy & Infectious Disease, Diagnostic Products Corporation, Los Angeles, CA. RATIONALE: Anti-CCD reactivity is known to exist in patient sera, resulting in false-positive immunoassay results, particularly with foods of plant origin. In this study, we evaluated the use of three specific IgE allergens_horseradish peroxidase (HRPO), ascorbate oxidase (ASO), and bromelain (BRO)_as immunoassay tools to detect anti-CCD reactivity. METHODS: Suspected CCD-reactive patient samples were selected on the basis of a negative case history to glycoprotein-containing foods and positive skin prick tests to a variety of pollen allergens. Specific IgE to the three allergens (HRPO, ASO, BRO) was determined using the IMMULITE® 2000 3gAllergy™ Specific IgE assay (Diagnostic Products Corporation, Los Angeles). Prior to analysis, standardized inhibition studies to confirm CCD reactivity were performed by preincubating patient sera with native and periodate-treated (chemically deglycosylated) allergen extracts (HRPO, ASO or BRO). RESULTS: In vitro studies of IgE sensitization to CCD were measured by comparing inhibition results between native and deglycosylated forms of HRPO, ASO, and BRO. All three are known to contain glycoprotein, although the exact number of glycan structures in each allergen varies. Greater than 90% inhibition was measured in each case, whereas negligible inhibition was measured for periodate-treated extracts. CONCLUSIONS: These data demonstrate convincingly that CCD-specific IgE interacted with the carbohydrate moieties of these glycoallergens. These glycoprotein-containing allergens, either together or individually, offer the opportunity to analyze discrepancies between clinical history, skin prick test results and in vitro results. Funding: Diagnostic Products Corporation
457
Bla g 6: A Calcium Binding German Cockroach Allergen Involved in Muscle Contraction J. Hindley1, S. Wünschmann1, L. D. Vailes1, S. M. Satinover2, J. A. Woodfolk2, M. D. Chapman1, A. Pomés1; 1R&D, INDOOR Biotechnologies, Inc., Charlottesville, VA, 2Allergy Division, University of Virginia, Charlottesville, VA. RATIONALE: The cockroach allergens that have been identified do not appear to account for the full repertoire of IgE responses. We investigated the importance of other Blattella germanica allergens that could contribute to cockroach allergy. METHODS: A B. germanica cDNA library was screened with a pool of sera from cockroach allergic patients. Three isoallergens were cloned, expressed in Pichia pastoris and purified by ion exchange and gel filtration chromatography. IgE responses to purified allergens were simultaneously measured in 104 sera using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. Homology modelling of the allergens was performed using Swiss-Model. RESULTS: Three isoallergens were identified and designated Bla g 6.0101, Bla g 6.0201 and Bla g 6.0301. The last two allergens shared 93 and 68% amino acid identity to Bla g 6.0101, respectively. The three isoallergens were 61-78% identical to insect troponins-C, 42-44% identical to vertebrate calmodulins, and contained two EF hand calcium binding domains. The prevalence of IgE binding to the recombinant isoallergens was ~15%. Calcium depletion by 10mM EGTA reduced IgE binding to Bla-g-6.0101 and Bla-g-6.0301 by 40-60%. In most cases, addition of 10mM CaCl2 increased IgE binding. No effect was seen for the controls Der-p-1 and Bla-g-1. Bla-g-6 models, based on the structures of vertebrate troponin-C, showed two structurally homologous lobes mainly constituted by -helices and loops and connected by a flexible linker. CONCLUSIONS: Bla g 6 is a minor cockroach allergen with a calcium dependent IgE reactivity, which may be involved in muscle contraction. Funding: INDOOR Biotechnologies, Inc.
458
Screening for Conformational Epitopes Using Heat Denaturation of Allergens on ELISA Plates B. Ning, R. M. Goldblum, D. M. Estes, M. A. Endsley, M. Watanabe, T. Midoro-Horiuti; Pediatrics, University TX Medical Branch, Galveston, TX. RATIONALE: Denatured, soluble allergens have been used to test for conformational epitopes, but the results may be difficult to interpret. A rapid and reproducible method for screening and classification of epitopes could be valuable in characterizing the fine specificity of the immune response to allergens. METHODS: After coating with purified Jun a 1, a major allergen of mountain cedar pollen, ELISA plates were heated at 37, 56 or 75C then tested with monoclonal antibodies (MAbs) to Jun a 1. RESULTS: A panel of 42 mouse IgG MAbs to Jun a 1 were assigned to four groups based on the sensitivity of their epitopes to heat. A single antibody recognized an epitope that was sensitive to 56C; 37 recognized epitopes that were partially sensitive to 56C; three were partially sensitive to 75C and one was resistant to even 75C. The epitope for a previously described MAb (KW-S91), which recognizes a linear epitope of Jun a 1 was resistant to 75C. One of the partially sensitive epitopes mapped to a conformation on the surface of Jun a 1. CONCLUSIONS: These data indicate that conformational epitopes are sensitive to heating the protein attached to plastic surfaces. Heat denaturation of allergens attached to ELISA plates is a simple, fast and reproducible method for screening the response to conformational epitope and preliminarily classifying MAbs. Funding: NIAID
459
SUNDAY
Allergic Reactions to Common Allergens may be Due to Evolutionary Immune Response to Conserved Domains (CDs) Present in Parasites and Allergens B. P. Bielory1, T. John2, L. Bielory3; 1University of Michigan, Ann Arbor, MI, 2UMDNJ - New Jersey Medical School, Newark, NJ, 3Medicine, Pediatrics & Ophthalmology, UMDNJ - New Jersey Medical School, Newark, NJ. RATIONALE: Since TH2 responses are elicited against helminthic infections and in atopic disorders, we have searched for common epitopes in protein sequences. METHODS: Using Entrez™ and BLAST™, NCBI a search and retrieval system for nucleotide/protein sequences to identify conserved protein domains between allergens and parasites (Schistosoma mansoni, Fasciola hepatica, Wuchereria bancrofti, Plasmodium falciparum, Babesia equi and Schistosoma japonicum) with matches having an E¬value equal to or <0 and S value closer to or 200. Over 1200 domains were examined. RESULTS: S.mansoni shares CDs with Can F3 (S=983,E=0; Canis familiaris), Fel D2 (S=879, E=0; Felis Catus). F.hepatica shares CDs with mite allergens Der F1 (S=144, E=4.2E-33) and Der P1 (S=133, E=6.1E30);), Blo T1 (S=145, E=1.3E-33; B.tropicalis). W.bancrofti shares CDs with Cla H4 (S=854, E=0; Cladosporium herbarum mold). S.japonicum shares CDs with IgE autoantigen Homo sapiens (S=196, E=4E-16) P. falciparum shares CDs with Bet VIII (S=69, E=7.9E-14; Betula pendulawhite birch) B. equi shares conserved domain with Der P1 (S=133, E=6.3E-30;). CONCLUSIONS: CDs exist in parasites and allergens supporting the theory that human immune response to common allergens is a direct consequence of constant stimulation of parasitic organisms generated by the evolution of the repertoire stored in the human genome over time. Funding: UMDNJ - New Jersey Medical School
456
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
Use of Cross-reacting Carbohydrate Determinants (CCD) in Specific IgE Detection U. BANIK; Product Support Allergy & Infectious Disease, Diagnostic Products Corporation, Los Angeles, CA. RATIONALE: Anti-CCD reactivity is known to exist in patient sera, resulting in false-positive immunoassay results, particularly with foods of plant origin. In this study, we evaluated the use of three specific IgE allergens_horseradish peroxidase (HRPO), ascorbate oxidase (ASO), and bromelain (BRO)_as immunoassay tools to detect anti-CCD reactivity. METHODS: Suspected CCD-reactive patient samples were selected on the basis of a negative case history to glycoprotein-containing foods and positive skin prick tests to a variety of pollen allergens. Specific IgE to the three allergens (HRPO, ASO, BRO) was determined using the IMMULITE® 2000 3gAllergy™ Specific IgE assay (Diagnostic Products Corporation, Los Angeles). Prior to analysis, standardized inhibition studies to confirm CCD reactivity were performed by preincubating patient sera with native and periodate-treated (chemically deglycosylated) allergen extracts (HRPO, ASO or BRO). RESULTS: In vitro studies of IgE sensitization to CCD were measured by comparing inhibition results between native and deglycosylated forms of HRPO, ASO, and BRO. All three are known to contain glycoprotein, although the exact number of glycan structures in each allergen varies. Greater than 90% inhibition was measured in each case, whereas negligible inhibition was measured for periodate-treated extracts. CONCLUSIONS: These data demonstrate convincingly that CCD-specific IgE interacted with the carbohydrate moieties of these glycoallergens. These glycoprotein-containing allergens, either together or individually, offer the opportunity to analyze discrepancies between clinical history, skin prick test results and in vitro results. Funding: Diagnostic Products Corporation
457
Bla g 6: A Calcium Binding German Cockroach Allergen Involved in Muscle Contraction J. Hindley1, S. Wünschmann1, L. D. Vailes1, S. M. Satinover2, J. A. Woodfolk2, M. D. Chapman1, A. Pomés1; 1R&D, INDOOR Biotechnologies, Inc., Charlottesville, VA, 2Allergy Division, University of Virginia, Charlottesville, VA. RATIONALE: The cockroach allergens that have been identified do not appear to account for the full repertoire of IgE responses. We investigated the importance of other Blattella germanica allergens that could contribute to cockroach allergy. METHODS: A B. germanica cDNA library was screened with a pool of sera from cockroach allergic patients. Three isoallergens were cloned, expressed in Pichia pastoris and purified by ion exchange and gel filtration chromatography. IgE responses to purified allergens were simultaneously measured in 104 sera using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. Homology modelling of the allergens was performed using Swiss-Model. RESULTS: Three isoallergens were identified and designated Bla g 6.0101, Bla g 6.0201 and Bla g 6.0301. The last two allergens shared 93 and 68% amino acid identity to Bla g 6.0101, respectively. The three isoallergens were 61-78% identical to insect troponins-C, 42-44% identical to vertebrate calmodulins, and contained two EF hand calcium binding domains. The prevalence of IgE binding to the recombinant isoallergens was ~15%. Calcium depletion by 10mM EGTA reduced IgE binding to Bla-g-6.0101 and Bla-g-6.0301 by 40-60%. In most cases, addition of 10mM CaCl2 increased IgE binding. No effect was seen for the controls Der-p-1 and Bla-g-1. Bla-g-6 models, based on the structures of vertebrate troponin-C, showed two structurally homologous lobes mainly constituted by -helices and loops and connected by a flexible linker. CONCLUSIONS: Bla g 6 is a minor cockroach allergen with a calcium dependent IgE reactivity, which may be involved in muscle contraction. Funding: INDOOR Biotechnologies, Inc.
458
Screening for Conformational Epitopes Using Heat Denaturation of Allergens on ELISA Plates B. Ning, R. M. Goldblum, D. M. Estes, M. A. Endsley, M. Watanabe, T. Midoro-Horiuti; Pediatrics, University TX Medical Branch, Galveston, TX. RATIONALE: Denatured, soluble allergens have been used to test for conformational epitopes, but the results may be difficult to interpret. A rapid and reproducible method for screening and classification of epitopes could be valuable in characterizing the fine specificity of the immune response to allergens. METHODS: After coating with purified Jun a 1, a major allergen of mountain cedar pollen, ELISA plates were heated at 37, 56 or 75C then tested with monoclonal antibodies (MAbs) to Jun a 1. RESULTS: A panel of 42 mouse IgG MAbs to Jun a 1 were assigned to four groups based on the sensitivity of their epitopes to heat. A single antibody recognized an epitope that was sensitive to 56C; 37 recognized epitopes that were partially sensitive to 56C; three were partially sensitive to 75C and one was resistant to even 75C. The epitope for a previously described MAb (KW-S91), which recognizes a linear epitope of Jun a 1 was resistant to 75C. One of the partially sensitive epitopes mapped to a conformation on the surface of Jun a 1. CONCLUSIONS: These data indicate that conformational epitopes are sensitive to heating the protein attached to plastic surfaces. Heat denaturation of allergens attached to ELISA plates is a simple, fast and reproducible method for screening the response to conformational epitope and preliminarily classifying MAbs. Funding: NIAID
459
SUNDAY
Allergic Reactions to Common Allergens may be Due to Evolutionary Immune Response to Conserved Domains (CDs) Present in Parasites and Allergens B. P. Bielory1, T. John2, L. Bielory3; 1University of Michigan, Ann Arbor, MI, 2UMDNJ - New Jersey Medical School, Newark, NJ, 3Medicine, Pediatrics & Ophthalmology, UMDNJ - New Jersey Medical School, Newark, NJ. RATIONALE: Since TH2 responses are elicited against helminthic infections and in atopic disorders, we have searched for common epitopes in protein sequences. METHODS: Using Entrez™ and BLAST™, NCBI a search and retrieval system for nucleotide/protein sequences to identify conserved protein domains between allergens and parasites (Schistosoma mansoni, Fasciola hepatica, Wuchereria bancrofti, Plasmodium falciparum, Babesia equi and Schistosoma japonicum) with matches having an E¬value equal to or <0 and S value closer to or 200. Over 1200 domains were examined. RESULTS: S.mansoni shares CDs with Can F3 (S=983,E=0; Canis familiaris), Fel D2 (S=879, E=0; Felis Catus). F.hepatica shares CDs with mite allergens Der F1 (S=144, E=4.2E-33) and Der P1 (S=133, E=6.1E30);), Blo T1 (S=145, E=1.3E-33; B.tropicalis). W.bancrofti shares CDs with Cla H4 (S=854, E=0; Cladosporium herbarum mold). S.japonicum shares CDs with IgE autoantigen Homo sapiens (S=196, E=4E-16) P. falciparum shares CDs with Bet VIII (S=69, E=7.9E-14; Betula pendulawhite birch) B. equi shares conserved domain with Der P1 (S=133, E=6.3E-30;). CONCLUSIONS: CDs exist in parasites and allergens supporting the theory that human immune response to common allergens is a direct consequence of constant stimulation of parasitic organisms generated by the evolution of the repertoire stored in the human genome over time. Funding: UMDNJ - New Jersey Medical School
456