Optimising extraction methods for multiplex detection of clinically relevant food allergens

Food allergy is a significant global issue, affecting up to 10% of the population. The requirement for accurate analytical methods for allergen detection are paramount in identifying allergen contamination and for dose level assessments in foods used for allergy diagnosis and immunotherapy.The detection of allergen protein in foods can be challenging, where food matrices and processing are known to impair extraction and quantification. Allergen extraction has been investigated previously; however fundamental knowledge gaps remain. Studies have not fully addressed how detection of component allergens (e.g. Cor a 9 from hazelnut) are impacted, presenting a challenge for implementation of allergen-specific detection methods. Additionally, data suggests several extraction methods may be required to extract and analyse each allergen source. Shared extraction buffers remain elusive and so limits the effectiveness of multiplex (multi-target) technology.Here, the extraction of 14 major food allergen proteins from multiple food matrices (source materials, incurred chocolate dessert, unbaked and baked biscuit matrices) reflecting different analytical challenges was investigated. These de novo materials were prepared specifically for the study and contained defined levels of allergenic protein. Ten extraction buffers were identified from previous internal and published research studies, and allergen content was determined using a fully quantitative allergen specific multiplex and ELISA assays.For optimal yield, different extraction buffers were required for incurred foods compared to source materials. Two extraction buffers provided strong recovery (typically 50-150%) of 14 allergenic proteins from complex incurred foods and most were readily detectable at 10 ppm (mg/kg), with scope for detecting considerably lower amounts. Yield of allergens were negatively impacted by baking, albeit to varying extent. Significant differences in detectable allergen were observed depending on baking temperature and sampling location within the biscuit; an important finding given that baked foods are commonly used in allergy diagnostic and therapeutic capacity.