Kidney Bean: A Major Sensitizer among Legumes in Asthma and Rhinitis Patients from India

BACKGROUND: The prevalence of IgE mediated food allergies has increased over the last two decades. Food allergy has been reported to be fatal in highly sensitive individuals. Legumes are important food allergens but their prevalence may vary among different populations. The present study identifies sensitization to common legumes among Indian population, characterizes allergens of kidney bean and establishes its cross reactivity with other legumes. METHODOLOGY: Patients (n = 355) with history of legume allergy were skin prick tested (SPT) with 10 legumes. Specific IgE (sIgE) and total IgE were estimated in sera by enzyme-linked immunosorbent assay. Characterization of kidney bean allergens and their cross reactivity was investigated by immunobiochemical methods. Identification of major allergens of kidney bean was carried out by mass spectrometry. PRINCIPAL FINDINGS: Kidney bean exhibited sensitization in 78 (22.0%) patients followed by chickpea 65 (18.0%) and peanut 53 (15%). SPT positive patients depicted significantly elevated sIgE levels against different legumes (r = 0.85, p<0.0001). Sera from 30 kidney bean sensitive individuals exhibited basophil histamine release (16-54%) which significantly correlated with their SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001). Kidney bean showed eight major allergens of 58, 50, 45, 42, 40, 37, 34 and 18 kDa on immunoblot and required 67.3±2.51 ng of homologous protein for 50% IgE inhibition. Inhibition assays revealed extensive cross reactivity among kidney bean, peanut, black gram and pigeon pea. nLC-MS/MS analysis identified four allergens of kidney bean showing significant matches with known proteins namely lectin (phytohemagglutinin), phaseolin, alpha-amylase inhibitor precursor and group 3 late embryogenesis abundant protein. CONCLUSION/SIGNIFICANCE: Among legumes, kidney bean followed by chick pea and peanut are the major allergic triggers in asthma and rhinitis patients in India. Kidney bean showed eight major allergens and cross reacted with other legumes. A combination of SPT, sIgE and histamine release assay is helpful in allergy diagnosis

Sensitization to legume allergens detected by sIgE estimation in ELISA.

<p>*Cut off value for sIgE positivity = 0.138 OD (≥3 times of control).</p

IgE ELISA inhibition of kidney bean extract using self protein as inhibitor.

<p>Kidney bean positive patients' pooled sera (1∶10 v/v) was preincubated with 5, 10, 50, 100, 1000, 10000 ng of three batches of kidney bean extract as inhibitors (Kb1, Kb2, Kb3), bromelain and CL (<i>Curvularia lunata</i>) were also used as inhibitor for IgE inhibition to kidney bean. ELISA was carried out on solid phase coated kidney bean (2 µg/100 µl/well) and preincubated sera.</p

Histamine release assay.

<p>Basophils in leukocyte suspension were stripped off the bound IgE. Stripped basophils were sensitized with sera of kidney bean hypersensitive patients. The sensitized basophils were stimulated with kidney bean extract. The Histamine released from 30 patients varied from 16% to 54% while in controls it is less than 6% (A). Scatter plot of the correlation analysis between % histamine release vs SPT (mm²) (B), % histamine release vs sIgE (C) and % histamine release vs total IgE (D) among 30 kidney bean sensitive individuals. Basophil histamine release has been found to be very well correlated with SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001) but not with total IgE (r =  −0.13, p = 0.4942).</p

IgE ELISA inhibition of kidney bean extract with self and heterologous legume extracts.

<p>For determination of cross reactive proteins between kidney bean and different legume extracts, 7 separate serum pools were prepared with sera of patients' skin test positive to kidney bean but negative to that of legume extract to be tested. Kidney bean positive patients' pooled sera (1∶10 v/v) was preincubated with 5, 10, 50, 100, 1000, 10000 ng of legume extracts namely black gram, chickpea, kidney bean, lentil, pea, peanut, soybean and pigeon pea as inhibitors. ELISA was carried out on solid phase coated kidney bean (2 µg/100 µl/well) and preincubated sera.</p

Sensitization to legume allergens identified by skin prick tests in patients of asthma, rhinitis or both.

<p>*p-values were calculated using Fisher's exact test.</p

Estimation of sIgE, total IgE and basophil histamine release among the 30 kidney bean sensitive (SPT +ve) patients and 5 controls (SPT -ve).

<p>Estimation of sIgE, total IgE and basophil histamine release among the 30 kidney bean sensitive (SPT +ve) patients and 5 controls (SPT -ve).</p

Demographic details of patients skin prick tested with different allergen extracts.

<p>Demographic details of patients skin prick tested with different allergen extracts.</p

Peptide mass fingerprint database search of selected spots from 1-DE of kidney bean proteins.

<p><b>Footnotes:</b> Abbreviations; Tht  =  Theoretical, Obs  =  Observed. ^aMatches a  =  Number of peptides matched with protein in MS/MS query. ^bSequence coverage b  =  Total percentage of proteins amino acid sequence covered by peptides in MS/MS analysis. ^cMASCOT score c =  >40 indicate identification or extensive homology (p<0.05).</p