Standardisation of allergen products: 4. Validation of a candidate European Pharmacopoeia standard method for quantification of major grass pollen allergen Phl p 5

• Published in: Allergy (Copenhagen) 2022-02, Vol.77 (2), p.633-642
• Main Authors: Zimmer, Julia, Schmidt, Sandra, Kaul, Susanne, Costanzo, Angèle, Buchheit, Karl‐Heinz, Brown, Shannon, Carnés, Jerónimo, Chapman, Martin, Chen, Aaron, De Neergaard, Michalade, Döring, Sascha, Hindley, James Phillip, Holzhauser, Thomas, Jorajuria, Sylvie, Le Tallec, David, Lombardero, Manuel, Iacovacci, Patrizia, Reese, Gerald, Sander, Ingrid, Smith, Bryan, Strecker, Daniel, Ree, Ronald, Zebina, Myriam, Vieths, Stefan
• Format: Article
• Language: English
• Subjects: Allergens; Allergens - chemistry; Allergies; Biological Standardisation Programme; ELISA; Enzyme-Linked Immunosorbent Assay; Humans; Laboratories; major allergen; Phl p 5; Phleum - chemistry; Plant Proteins - chemistry; Poaceae; Pollen; Reference Standards; Timothy grass; Variation
• ISSN: 0105-4538; 1398-9995
• DOI: 10.1111/all.15003

Background The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5‐specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. Methods A Phl p 5‐specific ELISA system was assessed with respect to accuracy, precision, inter‐assay (within laboratory) and inter‐laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. Results The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter‐assay variation (max. GCV 24%) and especially inter‐laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. Conclusions Based on the collaborative study results, the assessed Phl p 5‐specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method. We present the findings of an international ring trial on the validation of a Phl p 5‐specific ELISA system. The ELISA systems selected in the course of BSP090 will present the first examples of obligatory standard methods for allergen quantification in Europe. After implementation as standard methods, allergists will, for the first time, be able to compare marketed birch pollen and Timothy grass pollen allergen products based on major allergen content. Abbreviations: AIT, allergen immunotherapy; AUN, allergy units native; Bet v 1, Betula verrucosa allergen 1 (from birch pollen); BSP, Biological Standardisation Programme; CRS, chemical reference standard; DPP/ml, depigmented polymerized unit; ELISA, enzyme‐linked immunosorbent assay; IR, index of react