International Ring Trial Successful – ELISA Developed for Measurement of an Important Grass Allergen
14 / 2021
Therapy options for allergies are increasing in number. Various therapeutic allergen products are available for allergic patients, but there is a lack of standard methods for comparing the active ingredient content of the available products. In an international ring trial coordinated by the Paul-Ehrlich-Institut, a test method (ELISA) was trialled for the allergen Phl p 5 in timothy grass pollen, to which up to a quarter of the European population is sensitized. In all the participating laboratories, the method successfully quantified the allergen. The results are reported in Allergy in its online edition dated 9 July 2021.
This extensive ring trial involved 14 laboratories in Europe and the USA. The aim was to examine whether the test works in the different laboratories and whether the results are comparable within one laboratory but also between the different laboratories. Only test systems that successfully undergo this kind of ring trial are suitable for incorporation as standard methods in the European Pharmacopoeia. The ELISA validated here proved to be suitable for quantifying recombinant and natural (native) Phl p 5 with the necessary accuracy. Based on the results of this collaborative study, the specific immunoassay is to be proposed as a mandatory standard method for the European Pharmacopoeia.
Allergies affect huge numbers of people. As much as 10-25 percent of the population of the western world are sufferers of hay fever alone (allergic rhinitis or rhinoconjunctivitis; allergic inflammation of the nasal mucosa and/or the conjunctiva of the eye). In the case of allergies caused by grass pollens, 60 to 80 percent of sensitizations are due to the allergen Phl p 5 from timothy grass pollen (Phleum pratense).
A now well-established method for treating hay fever or insect allergies is de- or hyposensitization, whereby the patient is accustomed to the allergen through regular administration – initially in increasing doses – of the allergen itself, either orally, sublingually (under the tongue) or subcutaneously (under the skin). An important aspect for successful treatment is ensuring the quality of the allergen products.
The cornerstones of the quality requirements for allergen products are set out in the general monograph Allergen Preparations of the European Pharmacopoeia. The European Medicines Agency (EMA) has also specified guidelines on the quality and clinical development of allergen immunotherapy (AIT) products in order to further improve and harmonize the quality of products for AIT. As a result of this regulatory framework, the quality of AIT products has improved continuously over the last few years, but it is not yet possible to compare different products in relation to their active ingredients, since manufacturer-specific analysis methods for allergen content are still customary. For this reason, standard methods and corresponding allergen standards need to be established for cross-product comparability. The Biological Standardization Programme (BSP) project BSP090 by the European Directorate for the Quality of Medicines & HealthCare (EDQM) is aiming to establish reference standards and ELISAs for the quantification of Bet v 1, the major allergen in the pollen from birch, Betula verrucose, and Phl p 5, a primary allergen in the pollen of meadow timothy, Phleum pratense. The two recombinant reference standards were established and added to the monographic list on allergen preparations back in 2012. Inclusion of the Bet v ELISAs in the European Pharmacopoeia is also imminent.
In the international ring trial led by Prof. Stefan Vieths, Vice President of the Paul-Ehrlich-Institut, and scientists in the Allergology Division, a Phl p 5-specific ELISA was developed. ELISA stands for “Enzyme-Linked Immunosorbent Assay”. This involves an antibody-based detection process that detects and quantifies the Phl p 5 specifically in samples. The “sandwich ELISA” used here involves two antibodies that bind specifically with the antigen to be detected.
“Here at the Paul-Ehrlich-Institut, we have spent many years working to achieve high quality standards in allergen preparations. One important step was the introduction of the Therapy Allergen Ordinance in 2008. With the development and establishment of reference standards and standard methods, as here for the allergen Phl p 5, we are continuing our contribution to ensuring that allergy-sufferers can count on a growing number of high-quality therapeutic allergens,” comments Prof. Stefan Vieths, explaining the significance of these activities.