title: Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.
abstract: The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products.  The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.
authors: Paul-Ehrlich-Institut, Langen, Germany.
authors: S
authors: S
authors: Vieths
authors: D
authors: D
authors: Barber
authors: M
authors: M
authors: Chapman
authors: A
authors: A
authors: Costanzo
authors: A
authors: A
authors: Daas
authors: H
authors: H
authors: Fiebig
authors: K M
authors: KM
authors: Hanschmann
authors: M
authors: M
authors: Hrabina
authors: S
authors: S
authors: Kaul
authors: A
authors: A
authors: Ledesma
authors: P
authors: P
authors: Moingeon
authors: G
authors: G
authors: Reese
authors: C
authors: C
authors: Schörner
authors: R
authors: R
authors: van Ree
authors: B
authors: B
authors: Weber
authors: K H
authors: KH
authors: Buchheit
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001491:Basophils / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards*; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000592:standards*; D011058:Pollen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C076049:Phl p V protein, Phleum pratense; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula