Identification, purification and quantification of 20.2 kd IgE binding major allergen of aspergillus fumigatus for quality control

Author:

Vermani M 1; Vijayan VK 2; Agarwal MK 2

Author address:

1 Amity University Uttar Pradesh, Noida, India; 2 VP Chest Institute, Delhi, India

Full conference title:

European Academy of Allergy and Clinical Immunology Congress 2017

Date: 20 August 2020

Abstract:

Introduction: Airborne Aspergillus fumigatus is an important source of inhalant allergens. Current allergy diagnostic and therapeutic modalities employ crude non-standardized allergen extracts. Standardization of allergen extracts is crucial for improving their quality, key issue in diagnosis and therapy. We report a study on identification, purification and quantification of a 20.2 kd major allergen of A fumigatus to serve as a reference reagent for the standardization of crude extracts.

Objectives: Major allergens of A fumigatus were identified by performing immunoblots using sensitized patients’ sera. Purification of one major allergenic protein of crude extract was undertaken by subjecting it to salt precipitation, anion exchange column chromatography and fast protein liquid chromatography (FPLC).

Results: In immunoblots, 12 IgE binding proteins were detected, four being major allergens of A fumigatus- 90, 83, 34, 20.2 kd. The 20.2 kd component detected with maximum number of positive patients’ sera was purified to apparent homogeneity. The precipitate recovered on 80% ammonium sulfate precipitation of crude extract was subjected to anion exchange chromatography (DEAE-Sephacel) which yielded three fractions. The 20.2 kd component was detected in Afu-Fr1 with standard methods. Further, Afu-Fr1 was subjected to FPLC using a pre-packed mono-Q column. The eluates were pooled into five fractions. The 20.2 kd band was observed in Afu-F1a. Rechromatography of Afu-Fr1a again resulted in a single peak. At each step, allergenic activity was evaluated by EAST inhibition and immunoblots. For quantification of the purified allergen in crude extract, EAST inhibition assays with the sera pool of A fumigatus allergic patients were standardized. A dose-related inhibition was obtained in Afu-Fr1a EAST with Afu-Fr1a (purified protein) as well as the crude extract. Dose of purified protein and the crude extract required for 50% inhibition worked out to be 23.7 and 1599 ng, respectively. Thus, the concentration of purified protein (20.2 kd) in crude lyophilized A fumigatus extract worked out to be 14.8 lg/mg.

Conclusions: Thus, the 20.2 kd major allergen of A fumigatus has been purified to apparent homogeneity. The inhibition assays to quantify this purified allergen in crude extracts have been standardized. To ensure appropriate biopotency of crude A fumigatus extract, 20.2 kd protein may be used as a reference reagent for quality control.

Abstract Number: 0539

Link to conference website:

Link Conference abstract: 

EAACI 2017

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