Abstract
BackgroundLegumes are a rich source of proteins but are also potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity.MethodologyKidney bean allergen was purified using Q Sepharose column (anion exchanger) and eluates with high intensity were pooled to purify protein using Superdex 75 (gel filtration) and C18 column (RP-HPLC). Patients with history of kidney bean allergy were skin prick tested (SPT) with crude kidney bean extract and the purified protein. Specific IgE was estimated in sera by enzyme-linked immunosorbent assay (ELISA). Characterization of purified protein and its cross-reactivity was investigated by immunobiochemical methods. Identification of purified protein was carried out by tandem mass spectrometry.Principal FindingsPurified protein appeared as a single band at 31 kDa on SDS-PAGE and showed IgE binding to 88% patients’ sera by ELISA and immunoblotting. SPT with purified protein identified 78% hypersensitive patients of kidney bean. Significant release of histamine from sensitized basophils was observed after challenge with purified protein. PAS staining suggested it to be a glycoprotein, but no change in IgE binding was observed after periodate oxidation. The 31 kDa protein remained stable for 60 min on incubation with pepsin. The purified protein had high allergenic potential since it required only 102 ng of self protein for 50% IgE inhibition. Mass spectrometric analysis identified it as Phytohemagglutinin. It also showed hemagglutination with human RBCs. Cross-reactivity was observed with peanut and black gram with IC50 of 185 and 228 ng respectively.Conclusion/SignificanceA 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram.
Highlights
Legumes are rich source of proteins but are potential elicitor of IgE-mediated food allergy
It has been well documented that legumes such as kidney bean, peanut, chick pea, black gram pigeon pea, lentil are an important source of IgE mediated hypersensitivity [125]
Purification of 31 kDa Kidney Bean Protein The adsorbed proteins of kidney bean on Q Sepharose column were eluted using NaCl gradient and fractions were run on SDSPAGE
Summary
Legumes are rich source of proteins but are potential elicitor of IgE-mediated food allergy. It has been well documented that legumes such as kidney bean, peanut, chick pea, black gram pigeon pea, lentil are an important source of IgE mediated hypersensitivity [125]. Kidney bean has been identified as an emerging cause of food allergy among Indian population [2]. Kidney bean is consumed in Latin America, Africa, Middle East, Mediterranean area and India [127]. It contains high amounts of protein, abundant fibre and essential minerals like iron, zinc, calcium, and phosphorus [8]. Kidney bean has been identified as the major sensitizer among the atopic Indian population. Legumes are a rich source of proteins but are potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity
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