Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo)

Kumari, Dolly; Arora, Naveen; Kasera, Ramkrashan; Sridhara, S.; Kumar, Raj; Singh, Bhanu

doi:10.1016/j.imbio.2011.12.011

Cited by 15 publications

(9 citation statements)

References 45 publications

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“…The gel was incubated in 1% periodic acid in 3% acetic acid solution for 15 min and stained in Schiff’s reagent for 15 min in dark. After staining, the gel was washed with 5% sodium metabisulfite solution for 5 min followed by distilled water to visualize the protein bands [34].…”

Section: Methodsmentioning

confidence: 99%

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

Kasera

Singh

Lavasa³

et al. 2013

PLoS ONE

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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.

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Section: Methodsmentioning

confidence: 99%

“…The strip was washed with water and PBS and protein free sites were blocked with 3% defatted milk in PBS. The remaining steps of western blot were carried out as described earlier [34].…”

Section: Methodsmentioning

confidence: 99%

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

Kasera

Singh

Lavasa³

et al. 2013

PLoS ONE

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“…These allergens are generally resistance to heat and proteolytic enzymes 24 . Different chromatography and immuno-biochemical tools have been reported to purify and characterize the allergenic proteins from different food sources 25 . In the present study, we have purified PHA-L using a combination of approaches including fractionations, anion exchange chromatography, IgE immunoblotting and reverse phase-HPLC similar to the purification of other allergens [26][27][28] .…”

Section: Discussionmentioning

confidence: 99%

Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice

Kumar

Sharma

Das

et al. 2014

Immunopharmacology and Immunotoxicology

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Red kidney bean (Phaseolus vulgaris) is consumed worldwide as a vegetarian protein source. But, at the same time the allergenicity potential of red kidney bean is a matter of concern. This study is aimed towards purification, characterization, thermal stability, proteolytic digestion and allergenicity assessment of one of the clinically relevant allergens of red kidney bean. The purification of red kidney bean allergic protein was carried out with the help of column chromatography, IgE immunoblotting and reverse phase high-pressure liquid chromatography (RP-HPLC). The purified protein was characterized by peptide mass finger printing (PMF) and studied for its thermal stability, and proteolytic resistance using simulated gastric fluid (SGF) assay. The allergenicity potential of the purified protein was studied in BALB/c mice. The purified protein was identified as leucoagglutinating phytohemagglutinin (PHA-L) with molecular weight 29.5 kDa. The PHA-L showed resistance to heat as well as proteolytic enzyme. Higher levels of total IgE, specific IgE, and histamine were observed in PHA-L treated BALB/c mice when compared to control. Overall, PHA-L possesses characteristics of allergens and may play a potential role in the red kidney bean induced allergy.

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“…A number of IgE-reactive components of different molecular weight were detected in black gram seed, some of which retained IgE reactivity after heat treatment [105]. A 28-kDa major glycoprotein allergen was isolated and characterized from this legume seed, which displayed partial sequence homology with a Rho-specific inhibitor of transcription termination [106]. The degraded product (16 kDa) from the purified allergen after SGF (pepsin) treatment remained IgE reactive after 15 min of digestion.…”

Section: Molecular Allergology In Indiamentioning

confidence: 99%

Spectrum of Allergens and Allergen Biology in India

Bhattacharya

Sircar

Dasgupta³

et al. 2018

Int Arch Allergy Immunol

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The growing prevalence of allergy and asthma in India has become a major health concern with symptoms ranging from mild rhinitis to severe asthma and even life-threatening anaphylaxis. The “allergen repertoire” of this subcontinent is highly diverse due to the varied climate, flora, and food habits. The proper identification, purification, and molecular characterization of allergy-eliciting molecules are essential in order to facilitate an accurate diagnosis and to design immunotherapeutic vaccines. Although several reports on prevalent allergens are available, most of these studies were based on preliminary detection and identification of the allergens. Only a few of these allergen molecules have been characterized by recombinant technology and structural biology. The present review first describes the composition, distribution pattern, and natural sources of the predominant allergens in India along with the prevalence of sensitization to these allergens across the country. We go on to present a comprehensive report on the biochemical, immunological, and molecular information on the allergens reported so far from India. The review also covers the studies on allergy- related biosafety assessment of transgenic plants. Finally, we discuss the allergen-specific immunotherapy trials performed in India.

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Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo)

Cited by 15 publications

References 45 publications

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

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

Leucoagglutinating phytohemagglutinin: purification, characterization, proteolytic digestion and assessment for allergenicity potential in BALB/c mice

Spectrum of Allergens and Allergen Biology in India

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