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

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.

Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products.

Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 µg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.

The Central Nervous System Contains ILC1s That Differ From NK Cells in the Response to Inflammation.

Innate lymphoid cells (ILCs) are tissue resident cells with organ-specific properties. Here, we show that the central nervous system (CNS) encompasses ILCs. In particular, CD3-NK1.1+ cells present in the murine CNS comprise natural killer (NK) cells, ILC1s, intermediate ILC1s (intILC1s) and ex-ILC3s. We investigated the properties of CNS-ILC1s in comparison with CNS-NK cells during steady state and experimental autoimmune encephalomyelitis (EAE). ILC1s characteristically express CXCR3, CXCR6, DNAM-1, TRAIL, and CD200R and display heightened TNF-α production upon stimulation. In addition, ILC1s express perforin and are able to degranulate, although in a lesser extent than NK cells. Within the CNS compartments, ILC1s are enriched in the choroid plexus where very few NK cells are present, and also reside in the brain parenchyma and meninges. During EAE, ILC1s maintain stable IFN-γ and TNF-α levels while in NK cells the production of these cytokines increases as EAE progresses. Moreover, the amount of ILC1s and intILC1s increase in the parenchyma during EAE, but in contrast to NK cells, they show no signs of local proliferation. The upregulation in the inflamed brain of chemokines involved in ILC1 migration, such as CXCL9, CXCL10, and CXCL16 may lead to a recruitment of ILC1s from meninges or choroid plexus into the brain parenchyma. In sum, CNS-ILC1 phenotype, distribution and moderate inflammatory response during EAE suggest that they may act as gatekeepers involved in the control of neuroinflammation.