title: Quality control of house dust mite extracts by broad-spectrum profiling of allergen-related enzymatic activities.
abstract: BACKGROUND
  Diagnosis and immunotherapy of allergy against mites is based on complex extracts from large-scale cultures. However, the analysis of their composition using specific antibodies is limited. By taking advantage of the prevailing enzymatic nature of mite allergens, we have developed a broad-spectrum biochemical method for the standardization of native mite products.
  
  
  METHODS
  Microplate-based assays have been implemented for thirteen Dermatophagoides pteronyssinus enzymatic activities, associated with Der p 1, 3, 4, 6, 8, 9, 15 and 20 allergens. The dynamics of these activities along culture growth, and their profile in purified fractions (bodies and faeces) and international reference standards (WHO/IUIS, two CBER/FDA), have been characterized. The stability of enzymatic activities and major allergens under stress conditions (40°C) has been assessed in the presence/absence of specific protease inhibitors.
  
  
  RESULTS
  The analysis of enzymatic activities revealed distinct profiles along culture growth and between fractions (bodies and faeces). Remarkable differences were found when comparing international reference standards, being consistent with their source material (purified bodies or whole cultures). After 72 h at 40°C, only trypsin and alpha-amylase maintained high activity. Notably, the prominent role of trypsins in the hydrolytic degradation of major allergens is demonstrated by the use of inhibitors.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratorio de Interacción Planta-Insecto, Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas - CSIC, Madrid, Spain.
authors.forename: J C
authors.initials: JC
authors.lastname: Vidal-Quist
authors.affiliation: Laboratorio de Interacción Planta-Insecto, Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas - CSIC, Madrid, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Ortego
authors.affiliation: Laboratorio de Interacción Planta-Insecto, Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas - CSIC, Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Castañera
authors.affiliation: Laboratorio de Interacción Planta-Insecto, Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas - CSIC, Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Hernández-Crespo
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000276:immunology; D060829:Arthropod Proteins / Q000276:immunology / Q000592:standards; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000592:standards; D040002:Dermatophagoides pteronyssinus / Q000201:enzymology / Q000276:immunology; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis / Q000201:enzymology* / Q000276:immunology*; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards*; D039981:Pyroglyphidae / Q000201:enzymology* / Q000276:immunology*; D011786:Quality Control; D012015:Reference Standards; D013379:Substrate Specificity
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1
keywords: formulation; immunotherapy; mite physiology; proteases; stability