title: Purification and immunobiochemical characterization of a 31 kDa cross-reactive allergen from Phaseolus vulgaris (kidney bean).
abstract: BACKGROUND
  Legumes 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.
  
  
  METHODS
  Kidney 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.
  
  
  RESULTS
  Purified 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.
  
  
  CONCLUSIONS
  A 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram.
authors.affiliation: Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi, India.
authors.forename: Ramkrashan
authors.initials: R
authors.lastname: Kasera
authors.forename: Anand Bahadur
authors.initials: AB
authors.lastname: Singh
authors.forename: Shakuntala
authors.initials: S
authors.lastname: Lavasa
authors.forename: Komarla
authors.initials: K
authors.lastname: Nagendra
authors.forename: Naveen
authors.initials: N
authors.lastname: Arora
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D052179:Antigens, Plant / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D010367:Arachis / Q000276:immunology / Q000378:metabolism; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006636:Histamine Release / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D006967:Hypersensitivity / Q000097:blood / Q000276:immunology; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D013058:Mass Spectrometry; D008970:Molecular Weight; D010434:Pepsin A / Q000378:metabolism; D027805:Phaseolus / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010940:Plant Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D012882:Skin Tests
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; D007073:Immunoglobulin E; D010434:Pepsin A