Immunoinformatic epitope prediction to select monoclonal antibodies for Phl p 1 quantification.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Ole e 3, a Candidate for in vivo Diagnosis of Polcalcin Sensitization.

INTRODUCTION: Polcalcins belong to the family of calcium-binding proteins. They are ubiquitous in the plant kingdom and highly conserved, which leads to these panallergens showing a high degree of inter-cross-reactivity. They are responsible for allergic polysensitization, and therefore, their diagnosis is necessary for correct selection of immunotherapy. The objectives were to develop a method to purify native polcalcin with intact allergenic properties and to validate its use for diagnosis of polcalcin sensitization. METHODS: Ole e 3 was purified by immunoaffinity chromatography using anti-rChe a 3 polyclonal antibodies and identified by mass spectrometry. Calcium-binding assays were performed in immunoblot and ELISA assays. Diagnostic capacity of Ole e 3 was analyzed by ELISA and compared to ImmunoCAP with sera from a pollen-sensitized population. Cross-reactivity with other polcalcins was investigated by ImmunoCAP inhibition. RESULTS: Immunogenicity of purified Ole e 3 was not affected by the addition of calcium. However, the presence of a calcium chelator agent completely inhibited IgG binding by immunoblot and produced a 32.3% reduction in IgE binding by ELISA. Ole e 3 enabled diagnosis of polcalcin-sensitized patients, and a good correlation was revealed with ImmunoCAP. A 50% inhibition in IgE binding was obtained with 2.8 ng of Ole e 3 for rBet v 4 and 3.9 ng for rPhl p 7. DISCUSSION/CONCLUSION: Native Ole e 3 was purified by maintaining its allergenic properties. This innovative method enables obtaining this active native allergen to be used for in vivo diagnosis of polcalcin sensitization.