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The European Union CREATE Project: A model for international standardization of allergy diagnostics and vaccines

https://doi.org/10.1016/j.jaci.2008.07.030Get rights and content

Allergen measurements are used extensively in the formulation of allergy diagnostics and vaccines, yet no purified international allergen standards are available for calibration purposes. The aims of the European Union CREATE project were to develop international standards with verifiable allergen content. Purified natural and recombinant allergens were analyzed by means of SDS-PAGE, mass spectrometry, circular dichroism spectra, and small-angle x-ray scattering. IgE reactivity was assessed by means of direct RAST, RAST inhibition, immunoblotting, and basophil histamine release with sera from 961 allergic patients. Three recombinant allergens, rBet v 1, rPhl p 5a, and rDer p 2, were structurally indistinguishable from their natural counterparts and showed excellent IgE reactivity suitable for use as certified reference materials. A second tier of allergens (rPhl p 5b, rOle e1, rDer p 1, rDer f 1, and rDer f 2) was identified that could provide suitable candidates for certified reference materials with minor improvements to the recombinant proteins. Only rPhl p 1 was considered unsuitable as a reference material. Quantitative ELISAs were identified that accurately measured each allergen, except for rPhl p 1. The CREATE project has provided a major step forward in allergen standardization and provides a model for the development of a comprehensive panel of international reference preparations that will harmonize allergen measurements worldwide.

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Participating organizations and study design

The CREATE consortium comprised 28 organizations: 9 research laboratories, 11 clinical research groups, 6 allergen manufacturers, and 2 biotech companies from 9 European countries (Austria, Denmark, France, Germany, Italy, The Netherlands, Spain, Sweden, and the United Kingdom; see Table E1 in this article's Online Repository at www.jacionline.org). The consortium included 3 laboratories from governmental institutions involved in regulatory affairs: the Paul-Ehrlich Institute (Germany), the

Structural comparisons of purified natural and recombinant allergens

All allergens showed greater than 95% purity when analyzed by means of SDS-PAGE (Fig 2). The molecular weights of allergens produced in E coli showed good agreement with their natural counterparts. Mite allergens produced in P pastoris (rDer p 1 and rDer f 1) had significantly higher molecular weights because they were glycosylated (by using carbohydrates incorporated by P pastoris). Partial amino acid sequencing by means of ESI-QTOF-MS confirmed the identity of the allergens and accounted for

Discussion

The CREATE project had several successful outcomes. The project conducted a thorough structural comparison of purified natural and recombinant allergens and identified several recombinant allergens that are suitable as biologic reference preparations. CREATE provided a strong body of data on the IgE-binding activity of natural and recombinant allergens and has identified ELISA reagents that are suitable for allergen measurement. Three recombinant allergens were considered to be suitable as

A model for international allergen standardization

The CREATE project has provided a major step forward in allergen standardization and provides a coherent strategy for the future development of a comprehensive panel of international allergen standards. The criteria of allergen purity and IgE binding used in CREATE can readily be applied to other allergens. Improvements that have been made in the production of recombinant allergens since the CREATE project was initiated will make it easier to produce high-quality allergens with optimal IgE

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Supported in part by the 5th Framework Programme of the European Union (contract no. G6RD-CT-2001-00582) and by the following companies: ALK-Abelló, Allergopharma Joachim Ganzer KG, ASAC Pharmaceutical International SA, Biomay AG, Laboratorios Leti, HAL Allergy BV, Indoor Biotechnologies Ltd, and Stallergènes SA.

Disclosure of potential conflict of interest: M. D. Chapman is co-owner of Indoor Biotechnologies and has received research support from the National Institute for Environmental Health Sciences. F. Ferreira has received research support from Biomay and the Austrian Science Fund and has served as a consultant for Indoor Biotechnologies and the Allergen Online Database. M. Villalba has received research support from Ministerio de Ciencia y Tecnologia and ALK-Abelló. O. Cromwell is employed by Allergopharma Joachim Ganzer. M. Fernández-Rivas has received research support from the European Commission. S. Durham has received research support from GlaxoSmithKline and ALK-Abelló and has served as a member of the Immune Tolerance Network. S. Vieths has received honoraria from Phadia and the Food Allergy Resource and Research Program; is an associate for the Institute for Product Quality; has received research support from the European Union, the German Research Society, the Research Fund of the German Food Industry, Monsanto Company, and the European Directorate for the Quality of Medicines and Health Care; and has served as a member of the European Academy of Allergy and Clinical Immunology, the International Union of Immunological Societies, the European Agency for the Evaluation of Medicinal Products, the European Pharmacopeia Commission, the ILSI Health and Environmental Institute, the Protein Allergenicity Technical Committee, CEN, and Deutsche Gesellschaft fir Allergie und Klinische Immunologie. R. van Ree has consulting arrangements with Stallergènes, BIAL Aristegui, and HAL Allergy BV. The rest of the authors have declared that they have no conflict of interest.

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