This study is aimed to identify the allergens and determine their allergenic potency through immuno-biochemical methods from A. terreus, the most prevalent airborne allergenic fungus in West Bengal, India.
Proteins from a two-week old culture of A. terreus were extracted for determining its allergenicity by SPT, ELISA and histamine release assay. Protein profile was analyzed by 1D and 2D gel electrophoresis, followed by immunoblotting with 15 individual sensitized patients' sera and identification by mass spectrometry using MALDI-TOF/TOF. One of the major allergens, Triose phosphate isomerase (TPI) has been purified for bio-physical and biochemical studies, followed by in silico allergenicity assessment. The 3D structure of TPI was homology modelled by using crystal structure of TPI from Tenebrio molitor as template. Putative IgE binding peptides were synthesized and subsequently validated by in vitro experiments.
Histamine release, specific IgE, and skin sensitivity were found in higher amount in susceptible patients. 2D immunoblot revealed sixteen IgE reactive spots which were identified by MALDI-TOF/TOF. Amongst these, TPI was purified by two stage chromatography. It demonstrated a stable structure upon thermal denaturation using circular dichroism. Predicted IgE binding epitopes mapped on homology modelled structure showed that they were surface accessible. Synthetic epitope containing peptides showed significantly high IgE binding and histamine release with hypersensitive patients’ sera.
This finding unearthed novel allergens from A terreus and suggests that TPI is a major allergen with respect to immunoreactivity. Further, B cell peptides, identified by in silico methods can be potentially used in drug designing, allergy diagnosis and therapy.