Evaluation of Inhibitory Activity of Novel Monoclonal Antibodies Against Cat Allergen Fel d 7 and Their Application to Analyse Allergen Extracts.

Abstract

Current characterisation of cat dander/hair extracts used for allergy diagnosis or allergen-specific immunotherapy is mainly standardised towards the major allergen Fel d 1, while other allergens such as the lipocalin Fel d 7 are insufficiently characterised in such allergen products. This study aimed to produce recombinant Fel d 7 (rFel d 7) and murine IgG monoclonal antibodies (mAbs) specific to it for quantification in allergen extracts and assess the potential of the mAbs in inhibiting patients' IgE in functional assays. rFel d 7 was expressed in E. coli, purified by Ni-affinity and ion exchange chromatography, and physicochemically characterised by circular dichroism, Fourier-transform infrared spectroscopy, dynamic light scattering and mass spectrometry. Twenty hybridoma cell lines producing Fel d 7-specific mAbs were generated and sandwich ELISA was established for the quantitation of Fel d 7. Six different cat allergen extracts from different manufacturers and prepared from different sources were analysed and the concentration ranged from 0.02 μg/mg to 22.59 μg/mg. A mAb pool recognising non-overlapping epitopes inhibited the binding of human IgE-antigen complex formation (63.7% highest inhibition) and IgE-Fel d 7 cross-linking and consequent degranulation of basophilic cells (57.2% highest inhibition). We demonstrate the vast difference of Fel d 7 content in different cat allergen extracts, highlighting the necessity of improved standardisation of cat allergen extracts. The inhibition results showed that the analysed mAbs effectively inhibit rFel d 7 binding to human IgE, an assay we recommend for assessing the potency of allergen extracts as part of extract standardisation.