Quality control of house dust mite extracts by broad-spectrum profiling of allergen-related enzymatic activities

Abstract

Our method offers a robust approach to assess the complexity of mite extracts and highlights the critical importance of source materials for the composition and stability of finished products. The implementation of this approach in industry-based quality control procedures would contribute to the standardization of allergenic extracts used for diagnosis and immunotherapy.

Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite

“…However, the number of allergens that can be studied by this method is limited by the availability of specific antibodies. In addition, quantification may be influenced by polymorphism in allergen epitopes (van Ree, ) or may not detect partial degradation of the allergen (Vidal‐Quist et al ., ). Real‐time qPCR identifies changes in the expression of allergen genes without being subject to restriction in the number of genes (Vidal‐Quist et al ., ), although estimates of gene expression may not be directly related to allergen production if the gene is regulated by post‐transcriptional processes.…”

“…Real‐time qPCR identifies changes in the expression of allergen genes without being subject to restriction in the number of genes (Vidal‐Quist et al ., ), although estimates of gene expression may not be directly related to allergen production if the gene is regulated by post‐transcriptional processes. Finally, estimations of allergen‐related enzymatic activities allow the detection of changes in mite physiology that are directly or indirectly related to allergens, although this method is subject to the drawback that estimates may eventually be influenced by non‐allergenic proteins belonging to the same enzymatic family of the allergen (Vidal‐Quist et al ., ).…”

“…Der p 1 and Der p 2 allergens were quantified by sandwich ELISA using the Der p 1 ELISA kit (EL‐DP1A) and Mite Group 2 ELISA kit (EL‐D2) (Indoor Biotechnologies, Inc., Charlottesville, VA, U.S.A.). Nine enzymatic activities related to known allergens were estimated on mite extracts following previously described methods specifically designed for D. pteronyssinus (Vidal‐Quist et al ., ). Briefly, activities related to allergens Der p 1 (cysteine protease), Der p 3 (trypsin), Der p 6 (chymotrypsin), Der p 9 (elastase‐like) and Der p 15 (chitinase; both exo‐ and endo‐chitinase activities) were analysed using fluorometric assays, whereas Der p 4 (α‐amylase), Der p 8 (GST) and Der p 20 (arginine kinase) were estimated by colorimetric assays.…”

“…Improving the quality and consistency of HDM products would require both ensuring the reproducibility of the upstream process of production (Henmar et al ., ) by understanding the factors that determine the expression of allergens under rearing conditions, and implementing routine methods to monitor the complexity of API containing a whole set of clinically relevant allergens during downstream processes. In this regard, the present group has shown how the expression of several allergen genes is influenced by factors such as mite development, sex, temperature and relative humidity (RH) (Vidal‐Quist et al ., ) and has recently proposed a method for the quality control of native extracts by the analysis of allergen‐related enzymatic activities (Vidal‐Quist et al ., ). The enzymatic analysis conducted in D. pteronyssinus cultures at different ages, different fractions (mite bodies or faecal pellets), and on international D. pteronyssinus reference standards demonstrated how crucial the production process is to obtaining homogeneous allergen extracts.…”

Proteases of Dermatophagoides pteronyssinus

“…It also has been pointed out that enzymatic activity of allergens could lead to undesirable effects in immunotherapies, suggesting that functional deactivation may be useful in this treatment [12]. Also regarding immunotherapy, the protease activity of the extract has been proposed as a useful quality control measure for extract standardization [13]. Understanding the mechanisms whereby an active protease may influence sensitization and desensitization is still an active area of research, as evidenced by this special issue of International Journal of Molecular Sciences (IJMS).…”