Allergen quantification has become a relevant parameter for allergen extract characterization and to guarantee the consistency of the manufacturing process at allergen immunotherapy. The aim of this study was to develop and validate a method to quantify the major allergen Phl p 1 based on a prediction of the antigenic regions by immunoinformatic strategies.
Phl p 1 was purified from a Phleum pratense native extract by chromatographic methods. Immunoinformatic tools were used to predict B-cell epitopes. In silico predictions were verified by mapping linear epitopes with a peptide library and used to select the appropriate regions for producing the mAbs to develop an ELISA method, which was validated. Phl p 1 was quantified in 24 batches of P. pratense extracts.
Phl p 1 was purified with 95 % purity and completely functional. Eight B-cell epitopes in each of the two Phl p 1 isoforms were predicted. Two of the predicted B-cell epitopes overlapped with the experimentally determined peptides recognized by two mAbs selected for development of the kit. The quantification method demonstrated to be specific to Phl p 1, linear, accurate and precise in the range from 7.7 to 123.3 μg/mg. Mean Phl p 1 content was 28.95 μg of allergen/mg of lyophilized native extract and 44.23 μg of allergen/mg of lyophilized depigmented extract.
An ELISA method for measuring Phl p 1 in P. pratense extracts was developed and validated by producing the appropriate mAbs against epitopes selected by immunoinformatic tools.

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