Immunological In Vitro Assay for Quantification of Adjuvanted Allergoids
Year: 2025
Type: article
Abstract: BACKGROUND: Many IgE-mediated allergic disorders can be treated with allergen immunotherapy (AIT). In order to improve safety and efficacy, some AIT products contain allergen extracts which are chemically cross-linked to generate allergoids and are adsorbed to aluminum hydroxide adjuvant. The modification and adsorption impair accessibility of the protein and quantification of the allergoid content. METHODS: An ELISA-like assay to quantify the allergoid content in adjuvanted grass pollen allergoid AIT products (from here on called: AIT drug products; AIT-DPs) was developed using a fluorescence detection system. The high density of the aluminum hydroxide particles enabled pelleting the antigen complexes by centrifugation. Rabbit anti-grass pollen allergoid sera or a mouse anti-Phl p 5 monoclonal antibody (mAb) was used as the primary antibody. Protein content of the samples was quantified by nitrogen analysis. RESULTS: High specificity of the primary antibodies was confirmed by isoelectric focusing, gel-electrophoresis, and immunoblotting. Performance of the allergoid content assay was demonstrated in grass pollen AIT-DPs with high specificity and low/absent cross-reactivity with tree pollen or mite AIT-DPs. It was used to confirm batch-to-batch consistency and allergoid content of distinct grass pollen AIT-DPs. Overall, in relation to their total protein content, the allergoid content ranged between 0.8 and 2.1 relative to an in-house reference for all grass pollen AIT-DPs, whereas use of mAb revealed product-specific differences in the Phl p 5 amount. Additionally, the assay detects product alteration by heat stress. CONCLUSION: The described assay is suitable to quantify the allergoid content and quality of allergoids in complex with aluminum hydroxide. It is suitable for animal-free final product testing in vitro, for example, for batch release to ensure the quality of AIT-DPs.
Source: Allergy
DOI: 10.1111/all.16543
Institution: Paul Ehrlich Institut
Language: English
Subfield: Immunology and Allergy
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $5,300
OpenAlex ID: W4408997096