Denaturing Alternaria and Cladosporium Allergens

Denaturing Alternaria and Cladosporium Allergens

Abstracts AB147 J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2 576 578 577 579 Identifying Similar IgE-epitopes in Peanut and Walnut Allergens C. H...

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Abstracts AB147






Identifying Similar IgE-epitopes in Peanut and Walnut Allergens C. H. Schein1, S. J. Maleki2, S. Teuber3; 1University of Texas Medical Branch, Galveston, TX, 2USDA-ARS-SRRC, FPSQ, New Orleans, LA, 3 University of California, School of Medicine, Davis, CA. RATIONALE: About 35% of patients allergic to peanuts react to tree nuts. The physical property distance (PD) search tool of the Structural Database of Allergenic Proteins ( was used to identify meaningful similarities in the sequences that bind IgE in peanuts and walnuts allergens. METHODS: The PD tool found short sequence in walnut proteins that were similar (low PD score) to linear epitopes identified for the peanut allergens Ara h 1 and 2. Peptides were synthesized on ‘‘Spot membranes’’ which were reacted with IgE from patient sera, selected based on their reactivity to a major epitope of Ara h 2, and differing clinical reactivity to peanut, walnut, or both, confirmed with Western Blotting (WB) using extracts of both nuts, and basophil activation assays. Individual peptides were synthesized for Biacore and competitive Elisa tests with the same sera. The epitopes were visualized on models of the allergen 3D-structures. RESULTS: Several sequences were identified in the walnut allergen, Jug r 2, that reacted with patient IgE as well as the original peanut epitope, in sera from cross reactive individuals and those sensitive to walnuts. Cross-reactivity as determined from the spots was consistent with the reported clinical symptoms, but there was more recognition of the peptides than the whole protein (by WB). Peptides with a high PD value (lower similarity) were not recognized by any of the sera. CONCLUSIONS: The PD scale identifies areas in homologous allergens that are likely to be involved in cross-reactivity between food sources.

Structural Analysis of Der f 1 and Der p 1 Allergen-Antibody Complexes Explains Cross-reactivity of Group 1 Allergens M. Chruszcz1, M. D. Chapman2, L. D. Vailes2, A. Pome´s2, W. Minor1; 1 University of Virginia, Charlottesville, VA, 2INDOOR Biotechnologies, Inc., Charlottesville, VA. RATIONALE: Human IgE antibody responses to Group 1 allergens are strongly cross-reactive, whereas murine IgG antibody responses are largely species specific. In order to analyze the antigenic surface of these allergens, and investigate the basis of their cross-reactivity, we elucidated the crystal structures of Der f 1 and Der p 1 in complex with a cross-reacting monoclonal antibody (4C1). METHODS: X-ray diffraction analysis was used to investigate the molecular structures of Der f 1:4C1 and Der p 1:4C1 complexes, as well as structures of Der f 1, Der p 1 and 4C1 alone. RESULTS: Structural data reveal the epitope that is common to both Der f 1 and Der p 1. In both allergens the epitope is not only formed by the same amino acids, but their conformations are also the same. Moreover the amino acids forming the epitope have the same conformations whether complexed with antibody or not, in the case of both Der f 1 and Der p 1. The crystal structure of 4C1 alone shows that the CDR regions of the antibody do not significantly change in conformation upon allergen binding. CONCLUSIONS: The structures of these Group 1 allergens in complex with antibody revealed the specific amino acid residues involved in cross-reactivity and their interactions with the antibody. Such information, combined with site-directed mutagenesis studies, will facilitate identification of IgE binding epitopes and allows for the design of modified allergen molecules that could be used in recombinant vaccines for the treatment of dust mite allergy. Denaturing Alternaria and Cladosporium Allergens C. S. Barnes, F. Pacheco, J. M. Portnoy; Children’s Mercy Hospital, Kansas City, MO. RATIONALE: Fungal allergens are ubiquitous in the environment; however, there has been little progress in understanding their breakdown or disappearance. To investigate the denaturation of environmental allergenic material derived from Alternaria and Cladosporium using sodium hypochlorite both in vivo and in vitro, we conducted the following. METHODS: Freeze dried allergen extract from Alternaria alternata and Cladosporium herbarum was treated with hypochlorite solutions of concentrations up to 322 millimolar (mM). Remaining native allergenic material was then quantified using inhibition immunoassay, direct immunoassay and immunoassay with IgE from sensitized humans. Appropriate controls were employed to insure that the hypochlorite did not impact the assays themselves. RESULTS: The results of treating Alternaria or Cladosporium extract with solutions of sodium hypochlorite indicated that at concentrations greater than 100 mM the noticeable brown color of the extract was completely bleached away and at concentrations between 100 and 38 mM substantial bleaching was seen. All three immunoassay methods employed for determination of remaining antigenic and allergenic material revealed a general destruction of antigenic and allergenic material at concentrations of 38 mM or greater. At concentrations of 38 mM all human IgE recognized material was removed from the extract and from material grown on typical sheet rock. CONCLUSIONS: This work documents the ability of aqueous sodium hypochlorite solutions to denature fungal allergenic material from the common outdoor and indoor fungi Alternaria alternata and Cladosporium herbarum. This destruction of recognized antigenic and allergenic epitopes occurs at concentrations of hypochlorite commonly used for household cleaning.


Purification of Natural Bet v 2 by poly-L-Proline Affinity Chromatography and Preparative SDS-PAGE G. Reese1, B. Weber1, R. Valenta2, M. Stock1, C. Fritz1, H. Kahlert1, O. Cromwell1, H. Fiebig1; 1ALLERGOPHARMA Joachim Ganzer KG, Reinbek, GERMANY, 2Medical University Vienna, Vienna, AUSTRIA. RATIONALE: Even though recombinant allergens are frequently used for allergen characterization natural allergens are still the most relevant reference materials. However, using purified natural minor allergens (e.g. Bet v 2) containing minute amounts of major allergens (e.g. Bet v 1) as test antigen for component-resolved diagnosis may result in a high rate of false positive results. Spiking experiments indicated that a level as low as 0.001% of rBet v 1 in rBet v 2 preparations resulted in a high percentage of false positive results in subjectsÕ sera with IgE levels to birch with EAST classes 4 and higher. METHODS: Birch pollen extract was subjected to poly-L-Proline affinity chromatography to enrich Bet v 2, and preparative electrophoresis (PrepCell 491) to remove residual Bet v 1. Fractions were analyzed by ELISA, SDS-PAGE, and immunoblotting using subjectsÕ sera and Bet v 1 and Bet v 2-specific monoclonal antibodies. RESULTS: The Bet v 2 preparation analyzed after affinity chromatography bound IgE from all patientsÕ sera. Bet v 1-specific mAbs indicated the presence of approximately 0.01% of Bet v 1. Conventional SEC was not able to reduce the Bet v 1 level, fractionation by preparative electrophoresis, however, yielded a Bet v 2 preparation that contained less than 0.001% Bet v 1 reducing the rate of false positive IgE reactivities from 100% (24/24) to 8% (2/24) raising the assay specificity to 92%. CONCLUSIONS: Preparative electrophoresis in combination with other chromatography techniques is a useful method to obtain highly purified immunologically active allergens.