Purification and Immunobiochemical Characterization of a 31 kDa Cross-Reactive Allergen from Phaseolus vulgaris (Kidney Bean)

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.

Allergy to Peanut, Soybean, and Other Legumes: Recent Advances in Allergen Characterization, Stability to Processing and IgE Cross‐Reactivity

“…[28] Kasera et al demonstrated that a purified allergen of 31 kDa from green bean was stable to boiling for 60 min. [25] Similar heat stability to boiling has also been shown for pea allergens in several studies. [24,37]…”

“…In pea, Pis s ( Pisum sativum ) 3 was isolated and characterized as an ns‐LTP . In the case of green bean, several IgE‐binding proteins have been detected and partially characterized, but only an ns‐LTP from this legume has been described as a major allergen in a Spanish green bean‐allergic study population. The allergen has been completely characterized and named as Pha v ( Phaseolus vulgaris ) 3 .…”

Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

“…Histamine release upon rRhi o 1 challenge was performed and percentage of histamine release was calculated as described previously [ 22 ]. Briefly, PBMC’s (2 x 10 5 ) were isolated from healthy donors by HiSep TM LSM 1077 (HiMedia Laboratories, India) and bound IgE’s were stripped off by incubating in lactic acid buffer (pH 3.5) for 3 min.…”

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics

“…Another similar membrane was blotted with pooled sera from 2 non-atopic patients for control experiment. 21 …”

“…External calibration 21 was done using peptide calibration standard II mixture (Bruker Daltonics). Acquisition of spectra was done using Autoflex II MALDI-TOF/TOF machine in linear mode equipped with a pulsed nitrogen laser (λ=337 nm, 50 Hz) at 54% power in the positive ion mode.…”