Development, validation and stability study of an ELISA method to quantify the major allergen Jun a 1 in Juniperus...

A functional allergen quantification kit requires two key components: a pair of monoclonal antibodies (mAbs) that do not interfere with each other at the protein binding site, and a reference standard with sufficient purity and stability. Despite the clinical relevance of Jun a 1 in patients with Cupressaceae pollinosis, no commercial kits are currently available for its quantification in allergenic extracts. The aim of this study was to develop and validate a sandwich ELISA kit for the quantification of Jun a 1 in Juniperus ashei extracts, using appropriately selected mAbs and a stable standard. To this end, Jun a 1 was purified to >95% purity, as confirmed by SDS-PAGE, Western blot, and mass spectrometry analysis. Its stability was demonstrated after 48 months of storage. The linear B-cell epitopes recognized by the mAbs were identified and mapped onto the Jun a 1 structure, confirming that they are non-overlapping. Following validation, the ELISA method proved to be specific, linear, accurate, and precise. In conclusion, we successfully developed and validated an ELISA assay for the quantification of Jun a 1 in J. ashei extracts, supported by the generation of suitable monoclonal antibodies and a stable reference standard.