Immunological In Vitro Assay for Quantification of Adjuvanted Allergoids.

Area of Science:

• Immunology
• Biochemistry
• Analytical Chemistry

Background:

• Allergen immunotherapy (AIT) treats IgE-mediated allergic disorders.
• Modified allergens (allergoids) adsorbed to aluminum hydroxide improve AIT safety and efficacy.
• Chemical modification and adsorption hinder protein accessibility and quantification.

Purpose of the Study:

• Develop and validate an assay to quantify allergoid content in adjuvanted AIT drug products.
• Ensure batch-to-batch consistency and quality control for AIT products.

Main Methods:

• An ELISA-like assay with fluorescence detection was developed for adjuvanted grass pollen allergoid AIT products.
• Aluminum hydroxide particles were pelleted via centrifugation.
• Rabbit anti-allergoid sera or mouse anti-Phl p 5 monoclonal antibody (mAb) was used as the primary antibody.
• Nitrogen analysis quantified total protein content.

Main Results:

• The assay demonstrated high specificity for grass pollen allergoids, with minimal cross-reactivity to other allergens.
• Batch-to-batch consistency and allergoid content (0.8-2.1 relative to reference) were confirmed.
• Monoclonal antibody use revealed product-specific differences in Phl p 5 content.
• The assay detected product alterations induced by heat stress.

Conclusions:

• The developed assay reliably quantifies allergoid content and quality in aluminum hydroxide-adsorbed AIT products.
• This assay is suitable for animal-free in vitro final product testing and batch release.
• It ensures the consistent quality of AIT drug products for therapeutic use.