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A representative randomized group of 30 adult patients visiting the Allergology outpatient clinic at the University Medical Center Utrecht with a legume allergy for peanut, soybean, green pea, lupine, lentil or bean were included in the study. When this number was not reached, all available allergic patients were included. Selected patients were preferably diagnosed by a positive oral food challenge or by a convincing history combined with a positive immunoglobulin E (IgE) test in blood (>0.35 kU/L, ThermoFisher, Uppsala, Sweden). An overview of the details of the included patients can be found in Supplementary Table S1.

Non-processed and heat processed extracts and protein fractions from peanut (Arachis hypogaea), soybean (Glycine max), green pea (Pisum sativum), chickpea (Cicer arietinum), blue lupin (Lupineus angustifolius) and white lupine (Lupineus albus), black lentil (Lens culinaris) and green lentil (Lens culinaris puyensis), and faba bean (Vicia faba) and white bean (Phaseolus vulgaris).were made, as extensively described previously (17). The protein fractions, 7S/11S globulins (salt soluble globulins), 2S albumin (alcohol soluble prolamins), albumin (water soluble albumins) were extracted using the Osbourne extraction in a procedure adapted from Freitas et al. (18). In short, the globulin fraction was collected by solubilizing defatted legume meal with a high salt buffer (100 mM TRIS/HCl, 1 M NaCl, 10 mM EDTA and 10 mM EGTA, pH 8.2) and centrifuging. The supernatant was collected afterwards which contained the globulins that dissolved in the high salt buffer. By using 100 kDa ultra-filtration, the supernatant was divided in a 7S and 11S globulin fraction (the retentate) and the 2S albumin (the permeate) fraction. Individual seed storage proteins from peanut (Ara h 1, Ara h 2, Ara h 3, Ara h 6), soybean (Gly m 5 and Gly m 6), blue lupine (α-conglutin, δ-conglutin, and Lup an 1), green pea (pea albumin 1, pea albumin 2, Pis s 1 and legumin A), and white bean (phaseolin and legumin) were prepared as described in Smits et al. (17). Recombinant peanut allergen Ara h 7.0201 (Acc. no. B4XID4) was provided by EUROIMMUN.

The isolated extracts, protein fractions, and individual proteins were placed on a EUROLINE strip (EUROIMMUN, Lübeck, Germany) specially designed for this study by EUROIMMUN. Sensitization was assessed according to the standard manufacturer's instructions. The EUROLINE intensity units (EL) were evaluated using the EUROLineScan software and cross-reactive carbohydrate determinants (CCD) positive sera were reanalyzed after inhibition with Anti-CCD Absorbent (EUROIMMUN). An intensity of 3 (class 1) or higher was rated as positive.

20 µg of the 2S albumin and 7S/11S globulin fractions were diluted to 0.5 mg/ml in 100 mM Tris (pH 8.0) (40 µl in total). 8 µl of SDS 2.5% (final concentration 0.42% w/v) was added to the samples and the proteins were subjected to reduction, alkylation, tryptic digestion and strong cation exchange StageTip purification as previously described (19). Overnight proteolysis was achieved by adding 400 ng of trypsin per 20 µg of sample (E:S ratio of 1:50). The albumin fractions were processed in a similar way but were diluted to 0.5 mg/mL during the first step in HPLC-grade water instead of 100 mM Tris (pH 8.0) and 500 mM Tris was added to the 2.5% SDS solution that was added to the samples before reduction with DTT. NanoLC-MS/MS analysis was performed on an Easy LC 1,000 nanoscale liquid chromatography system (Thermo Fisher Scientific) coupled to a Q-Exactive mass spectrometer (Thermo Scientific) as previously described with small changes (20). Four microliters (100 ng) of the protein fractions were loaded at a 500 nl/min flow rate onto the analytical column, and peptides were eluted through the reversed-phase column via a 45-minute linear gradient. The MS data were processed using Proteome Discoverer v.1.4 (Thermo Scientific) and SEQUEST as search engine. Proteins were identified by searching the mass spectrometric data against the Uniprot Fabaceae database (434,155 entries) accessed on September 2017. Protein hits based on 2 successful peptide identifications (filtered by Percolator, FDR < 0.01) were considered valid.

15 µl (1 µg/ml) of extract or protein fraction was mixed with 5 µl of 4 × Laemmli sample buffer (BioRad, Hercules, CA) supplemented with DTT. The samples were heated for 5 min at 95°C and shortly vortexed afterwards. 5 µl of Precision Plus Protein Dual Color Standards marker (BioRad, Hercules, CA) and 10 µl of the protein sample were loaded into the wells of an Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gel (BioRad, Hercules, CA) in a buffer tank (Bio-Rad Laboratories Mini-PROTEAN Tetra Cell™) filled with 1 × TGS (BioRad, Hercules, CA). The gels were stained overnight on a plate shaker with InstantBlue™ (Coomassie) gel staining (Expedion, United Kingdom) and then washed with demineralized water for at least 1 h and an image of the gel was made (ChemiDoc™ MP).

A subpopulation of peanut, green pea and lentil-allergic subjects was selected for the inhibition assays. To study the capacity of different 2S albumin and 7S/11S fractions to inhibit IgE binding, sera from selected patients were pre-incubated with unprocessed 2S albumin or 7S/11S fractions of peanut, green pea and lentil. Briefly, 1, 10 and 100 µg/ml of the respective fractions were added to 1:10 (EUROLINE washing buffer) diluted sera and incubated at room temperature for 30 min on an orbital shaker (300 rpm). Subsequently, a line blot was performed. The same protein extracts where used for preparing the line blots and performing the inhibition assay. Pre-incubation with cow's milk extract served as negative control. The positive controls comprised of pre-incubation with the same fraction IgE (self-inhibition) and patients who did not show self-inhibition were excluded from analysis. Inhibition was calculated as percentage with respect to the not inhibited measurement.

Co-sensitization occurs when different IgE antibodies are produced by the patient that bind to proteins but are not necessarily targeted at common structural features (21). In contrast, cross-reactive IgE binding is characterized by IgE antibodies that bind structurally homologous proteins that share common epitopes and cross-reactivity can specifically be determined by inhibition assays (21). Likewise, co-allergy is defined by the presence of clinical allergic complaints for two or more sources, and cross-allergy is defined as the presence of clinical allergic complaints elicited by cross-reactive IgE binding to multiple sources.

Descriptive analyses were performed to report the frequency of (co-)sensitization and (co-)allergy using SPSS Statistics 25 (IBM Corporation, Armonk, NY, United States). Additionally, the odds ratios (OR) were calculated after adjustment by the Haldane correction to assess the association between legume sensitization and allergy. The Basic Local Alignment Search Tool (BLASTP) was used to investigate the percentage identity. Matches of greater than 50% identity indicated potential cross-reactivity (22).

The frequency of sensitization for 10 different legumes (peanut, soybean, green pea, chickpea, blue/white lupine, black/green lentil, faba/white bean) in six legume-allergic patient groups (peanut n = 30, soybean n = 30, green pea n = 30, lupine n = 30, lentil n = 17 and bean n = 9) is shown in Table 1. In all 6 legume-allergic patients groups, co-sensitization was seen for each of the tested legume with a percentage of at least 36.7%. In the bean-allergic patient group, the frequency of co-sensitization for other legumes was the highest (between 77.8% and 100%), while in the soybean and peanut-allergic patient groups lower rates were found (36.7%–76.7%). The percentages in Table 1 indicated that when patients were allergic to green pea, lupine, lentil or bean, they were most likely sensitized to other legumes as well (ranging from 58.8%–100%). In contrast, patients allergic to peanut or soybean were less likely sensitized to other legumes (36.7%–76.7%).

Individual sensitization profiles of the six legume-allergic patient groups to various legume protein extracts are provided in Figure 1. The allergic symptoms for each individual are displayed in Figure 1A. A limited degree of mono-sensitization was found in the soybean (16.7%), peanut (10%), and green pea-allergic (3.3%) patient groups. Co-sensitization for all 10 legumes was frequently seen in all legume-allergic patient groups (peanut (23.3%), soybean (33.3%), green pea (50%), lupine (43.3%), lentil (41.2%), and bean (66.7%) (Supplementary Table S3 and Figure 1B)). Remarkably, co-sensitization for less than six legumes was not seen for lupine, lentil, and bean-allergic patients as opposed to peanut, soybean and green pea-allergic patient groups. This indicates that lupine, lentil, and bean-allergic patients were always sensitized to other legumes as well, while peanut and soybean-allergic patients were not. A substantial percentage (20%) of soybean-allergic patients showed no IgE binding to one of the tested soybean extracts. This is most likely caused by the absence of the PR10-protein Gly m 4, an important allergen in soymilk, in the extracts (Supplementary Table S3).

IgE binding for different protein fractions was further evaluated by heat maps in Figure 1C (7S/11S globulins), Figure 1D (2S albumins), and Figure 1E (albumins). A high frequency of co-sensitization between the 7S/11S globulin fractions of the 10 legumes was observed, with IgE binding intensity profiles that resemble the sensitization profile as shown in Figure 1B (any extract, fraction or component). The mean intensity of IgE binding for each of the tested legumes was consistently higher for the 7S/11S globulin fraction compared to the 2S albumin and albumin fraction, except for the peanut 2S albumin fraction. Co-sensitization between 2S albumin fractions was more frequently seen compared to the albumin fractions. The most notable co-sensitization was seen for 2S albumin fraction of peanut and green lentil. 66.7% of the peanut-allergic patients, sensitized to the peanut 2S albumin fraction, were also sensitized to 2S albumin fraction of green lentil. Vice versa, 41.2% of the lentil-allergic patients were also sensitized to the 2S albumin fraction from peanut. The frequency of sensitization for the albumin fraction was in general low, except for peanut.

The frequency of co-sensitization between individual 7S globulins (Figure 2A), 11S globulins (Figure 2B), and 2S albumins (Figure 2C) was further explored for individual legume proteins. Co-sensitization for individual 7S and 11S globulins was highly prevalent in the soybean, green pea, lupine, lentil, and bean-allergic patient groups, which is consistent with the high co-sensitization between the 7S/11S globulin fractions of the various legumes. Interestingly, co-sensitization for the 7S and 11S globulins was in general frequently seen, with exception to phaseolin and legumin from white bean. The frequency of sensitization for these proteins was low, even in the bean-allergic patient group. Co-sensitization between the peanut 2S albumins and δ-conglutin from blue lupine was more frequently seen than between peanut and pea albumin 1, especially in the lupine-allergic patient group. Co-sensitization between the pea albumin 2, the peanut allergens, and blue lupin δ-conglutin was observed. 5 out of 7 green pea-allergic patients who were sensitized to pea albumin 1 were mono-sensitized to this 2S albumin. Together, the data indicate that co-sensitization is mainly seen between 7S and 11S globulins and to a lesser extent between 2S albumins.

In peanut and soybean-allergic patients, co-allergies for green pea, lupine, lentil and bean were uncommon (≤16,7%), while in green pea, lupine, lentil, and bean-allergic patients a peanut (64.7%–77.8%) or soybean (50%–64.7%) co-allergy was frequently seen (Figure 3, Table 2). A co-allergy for bean (0%–35.3%) was the least common co-allergy in all groups. Conversely, co-allergies to most of the other legumes were frequently (55.6%–77.8%) seen in the bean-allergic group. Furthermore, it is interesting to note that in soybean-allergic patients the frequency of a peanut (63.3%) co-allergy was much higher compared to co-allergy to other legumes (3.3%–16.7%). In all legume-allergic groups, the frequency of co-allergy was less prevalent compared to the frequency of co-sensitization. This indicates that a considerable part of the observed co-sensitizations may be not clinically relevant. The clinical relevance of the sensitization for different protein fractions (Figures 3C–E) is complex to interpret. We therefore calculated the OR for clinical relevant food allergy when sensitized to 7S/11S globulin or 2S albumin fractions. (Table 2). Sensitization for the 7S/11S globulin or the 2S albumin fraction from peanut significantly increased the risk for a peanut co-allergy in soybean, green pea, lupine, and lentil-allergic patients compared to patients that were not sensitized to the 7S/11S globulin or 2S albumin fraction from peanut (OR ranging from 12.89 [95% CI 1.97–84.12] to 94.71 [95% CI 4.39–2041.8]). Peanut and soybean-allergic patients that were sensitized to the 2S albumin fraction from lupine, had a significantly higher chance to have a lupine co-allergy compared to patients that were not sensitized to 2S albumin fraction from lupine (OR 11.18, 95% CI 1.41–88.95 for peanut as well as soybean). However, most of the OR were not statistically significant, indicating that co-sensitization often is not associated with a clinically relevant co-allergy. In conclusion, co-sensitization for 7S/11S globulin or 2S albumin fractions from peanut is associated with clinically relevant co-allergy for peanut in almost all legume-allergic patient groups, whereas co-sensitization for 7S/11S or 2S albumin fractions from other legumes is clinically less relevant.

Inhibition assays were performed as a case study to investigate whether (and to which extent) clinically relevant and irrelevant co-sensitization was caused by cross-reactivity in subpopulation of peanut, green pea and lentil-allergic subjects (Supplementary Table S2, Supplementary Figure S1). Potential cross-reactivity between peanut and lentil fractions (2S albumin and 7S/11S) was examined by cross-inhibition of IgE binding using sera from peanut-allergic and lentil-allergic subjects (Figure 4A, Supplementary Figure S2). An inhibitor concentration of 10 µg/ml was used as a negative control as 100 µg/ml showed inhibition for serum 3 (indicated with a black cross). Pre-incubation with peanut 2S or 7S/11S fractions resulted in some inhibition of IgE binding to the respective lentil fractions (median inhibition 2S albumin: 11%, median inhibition 7S/11S globulin: 67%). Vice versa, no inhibition or only limited inhibition of IgE binding to the 2S albumin and 7S/11S peanut fractions was observed upon pre-incubation with the respective lentil fractions. These observations indicate that sensitization for peanut fractions is, to a limited extent, responsible for co-sensitization for lentil fractions. However, co-sensitization between lentil and peanut is not caused by primary sensitization for lentil fractions. In contrast to peanut-allergic subjects, pre-incubation with lentil and green pea 2S albumin or 7S/11S fractions resulted in an inhibition (79% to 90%) of IgE binding in lentil and pea-allergic subjects (Figure 4B). Thus, clinically relevant co-sensitization between green pea and lentil in subjects co-allergic to green pea and lentil can to a large extent be explained by cross-reactivity.