Alanine Scanning of the Unstructured Region of Ara h 2 and of a Related Mimotope Reveals Critical Amino Acids for IgE Binding.

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.

Ara h 2 Peptide Mix Improves the Diagnosis of Peanut Allergy and Is Relevant for Ara h 2-Induced Mast Cell Activation.

BACKGROUND: A precise diagnosis of peanut allergy is extremely important. We identified 4 Ara h 2 peptides that improved Ara h 2-specific IgE (sIgE) diagnostic accuracy. OBJECTIVE: To assess the diagnostic utility of sIgE to the mixture of these peptides and their role in mast cell response to peanut allergens. METHODS: sIgE to the peptide mix was determined using ImmunoCAP. Its diagnostic utility was compared with Ara h 2-sIgE and sIgE to the individual peptides. The functional relevance of the peptides was tested on the mast cell activation test using laboratory of allergic diseases 2 cell line and flow cytometry. RESULTS: A total of 52 peanut-allergic (PA), 36 peanut-sensitized but tolerant, and 9 nonsensitized nonallergic children were studied. Peptide mix-sIgE improved the diagnostic performance of Ara h 2-sIgE compared with Ara h 2-sIgE alone (area under the receiver operating characteristic curve .92 vs .89, respectively; P = .056). The sensitivity and specificity of Ara h 2-sIgE combined with the peptide mix were 85% and 96%, respectively. sIgE to individual peptides had the highest specificity (91%-96%) but the lowest sensitivity (10%-52%) compared with Ara h 2-sIgE (69% specificity and 87% sensitivity) or with peptide mix-sIgE (82% specificity and 63% sensitivity). Peptide 3 directly induced mast cell activation, and the peptide mix inhibited Ara h 2-induced activation of mast cells sensitized with plasma from Ara h 2-positive PA patients. CONCLUSIONS: sIgE to the peptide mix improved the diagnostic performance of Ara h 2-sIgE similarly to sIgE to individual peptides. The peptides interfered with Ara h 2-induced mast cell activation, confirming its relevance in peanut allergy.

IgE Recognition and Structural Analysis of Disulfide Bond Rearrangement and Chemical Modifications in Allergen Aggregations in Roasted Peanuts.

Given that roasting changes the structure and allergenicity of peanut allergens, the structural information of peanut allergens must be expounded to explain the alteration in their allergenicity. This work focused on allergen aggregations (AAs) in roasted peanuts. IgE recognition capability was assessed via western blot analysis. The disulfide bond (DB) rearrangement and chemical modification in AAs were identified by combining mass spectroscopy and software tools, and structural changes induced by cross-links were displayed by molecular dynamics and PyMOL software. Results showed that AAs were strongly recognized by IgE and cross-linked mainly by DBs. The types of DB rearrangement in AAs included interprotein (98 peptide pairs), intraprotein (22 peptide pairs), and loop-linked (6 peptides) DBs. Among allergens, Ara h 2 and Ara h 6 presented the most cysteine residues to cross-linkf with others or themselves. DB rearrangement involved IgE epitopes and induced structural changes. Ara h 1 and Ara h 3 were predominantly chemically modified. Moreover, chemical modification altered the local structures of proteins, which may change the allergenic potential of allergens.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens.

BACKGROUND: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. OBJECTIVE: We investigated the unexpected cross-reactivity between peanut major allergens. METHODS: Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. RESULTS: Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. CONCLUSION: True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

IgE Epitope Profiling for Allergy Diagnosis and Therapy - Parallel Analysis of a Multitude of Potential Linear Epitopes Using a High Throughput Screening Platform.

Immunoglobulin E (IgE) is pivotal for manifestation and persistence of most immediate-type allergies and some asthma phenotypes. Consequently, IgE represents a crucial target for both, diagnostic purposes as well as therapeutic approaches. In fact, allergen-specific immunotherapy - aiming to re-route an IgE-based inflammatory response into an innocuous immune reaction against the allergen - is the only curative approach for IgE-mediated allergic diseases known so far. However, this requires the cognate allergen to be known. Unfortunately, even in well-characterized allergics or asthmatics, often just a small fraction of total IgE can be assigned to specific target allergens. To overcome this knowledge gap, we have devised an analytical platform for unbiased IgE target epitope detection. The system relies on chemically produced random peptide libraries immobilized on polystyrene beads ("one-bead-one-compound (OBOC) libraries") capable to present millions of different peptide motifs simultaneously to immunoglobulins from biological samples. Beads binding IgE are highlighted with a fluorophore-labeled anti-IgE antibody allowing fluorescence-based detection and isolation of positives, which then can be characterized by peptide sequencing. Setting-up this platform required an elaborate optimization process including proper choice of background suppressants, secondary antibody and fluorophore label as well as incubation conditions. For optimal performance our procedure involves a sophisticated pre-adsorption step to eliminate beads that react nonspecifically with anti-IgE secondary antibodies. This step turned out to be important for minimizing detection of "false positive" motifs that otherwise would erroneously be classified as IgE epitopes. In validation studies we were able to retrieve artificial test-peptide beads spiked into our library by using IgE directed against those test-peptides at physiological concentrations (≤20 IU/ml of specific IgE), and disease-relevant bead-bound epitopes of the major peanut allergen Ara h 2 by screening with sera from peanut allergics. Thus, we established a platform with which one can find and validate new immunoglobulin targets using patient material which displays a largely unknown immunoglobulin repertoire.

The forgotten 2S albumin proteins: Importance, structure, and biotechnological application in agriculture and human health.

2S albumin proteins are a group of important seed storage proteins (SSPs) essential to seeds at early and late developmental stages, by providing amino acids and other nutrients during germination and for seed defense. 2S albumins possess a well-conserved cysteine supporting the stability of temperature, pH, and proteolysis. The 3D structure rich in alpha-helices and positively charged is particularly suited for antibacterial and antifungal activity, which is presented by many 2S albumins. However, the hypervariable region present in 2S albumins induces allergenic reactions. Because of that, 2S albumins have never been recognized for their biotechnological potential. However, the development of servers used for the rational design of antimicrobial molecules has now brought a new application to 2S albumins, acting as a model to design antimicrobial molecules without the toxic or allergenic effects of 2S albumins. Therefore, this review is focused on discussing the importance of 2S albumins to seed development and defense and the biochemical, structural and functional properties of these proteins thought to play a role in their antimicrobial activity. Additionally, the application of 2S albumins to design synthetic antimicrobial peptides is discussed, potentially bringing new functions to these forgotten proteins.

Effect of pH regulation on the components and functional properties of proteins isolated from cold-pressed rapeseed meal through alkaline extraction and acid precipitation.

Cold-pressed rapeseed meal with high protein content (38.76% protein dry weight basis) was used to prepare rapeseed protein isolates (RPIs) by alkaline extraction (pH 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0) and acid precipitation (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5). The protein with an intact structure and the highest yield (65.08%) was obtained at extraction pH 9.0 and precipitation pH 4.5, accompanied by the lowest D-amino acid content, the lightest colour and the lowest contents of glucosinolates (2.85 mmol/kg), phytic acid (1.05 mg/g) and sinapine (0.68 mg/g). Additionally, water/oil absorption, foaming and emulsifying capacities decreased with decreasing precipitation pH, while the solubility showed the reverse trend. During gastric simulation digestion, the α-polypeptide of cruciferin and napin in the RPIs showed digestive resistance. Overall, pH regulation might be an effective method to isolate high quality RPIs for use in the food processing industry.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Polyphenol-oxidase-catalyzed cross-linking of Ara h 2: reaction sites and effect on structure and allergenicity.

BACKGROUND: Peanut is among the most common of food allergies, and one of its allergens is Ara h 2. A previous study revealed that this allergen was recognized by serum immunoglobulin E (IgE) in over 90% of a peanut-allergic patient population. Enzymatic cross-linking is a popular processing method used to tailor food functionality, such as antigenicity. RESULT: The cross-linking reactions of Ara h 2 were catalyzed by polyphenol oxidase (PPO), and the relevant reaction sites were identified using mass spectrometry and StavroX software. Two pairs of intramolecular cross-linking peptides and two intermolecular cross-linking peptides were found. Intramolecular cross-linking was speculated to occur between ARG131 (amino acids 116-131) and TYR65 (amino acids 63-80) and between TYR60 (amino acids 56-62) and ARG92 (amino acids 92-102); the intermolecular cross-linking sites were ARG31 with TYR84 or TYR89 and TYR65 or TYR72 with ARG92 or ARG102 . Three out of four cross-linking peptides were found in α-helices, and destruction of this secondary structure resulted in a loose tertiary structure. Although seven linear allergen epitopes were involved in cross-linking, the IgE binding capacity of protein changed slightly, while its sensitization potential decreased in mouse model. CONCLUSION: Exploring the structural change of Ara h 2 after cross-linking is beneficial in further understanding the influence of structure on sensitization. This result indicated the future possibility of precision processing on structure of proteins to improve their properties. © 2019 Society of Chemical Industry.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.

Identification and Quantification of DPP-IV-Inhibitory Peptides from Hydrolyzed-Rapeseed-Protein-Derived Napin with Analysis of the Interactions between Key Residues and Protein Domains.

Previously reported peptides derived from napin of rapeseed ( Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IV-inhibitory activity (IC50 = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.

Binding of peanut allergen Ara h 2 with Vaccinium fruit polyphenols.

The potential for 42 different polyphenols found in Vaccinium fruits to bind to peanut allergen Ara h 2 and inhibit IgE binding epitopes was investigated using cheminformatics techniques. Out of 12 predicted binders, delphinidin-3-glucoside, cyanidin-3-glucoside, procyanidin C1, and chlorogenic acid were further evaluated in vitro. Circular dichroism, UV-Vis spectroscopy, and immunoblotting determined their capacity to (i) bind to Ara h 2, (ii) induce protein secondary structural changes, and (iii) inhibit IgE binding epitopes. UV-Vis spectroscopy clearly indicated that procyanidin C1 and chlorogenic acid interacted with Ara h 2, and circular dichroism results suggested that interactions with these polyphenols resulted in changes to Ara h 2 secondary structures. Immunoblotting showed that procyanidin C1 and chlorogenic acid bound to Ara h 2 significantly decreased the IgE binding capacity by 37% and 50%, respectively. These results suggest that certain polyphenols can inhibit IgE recognition of Ara h 2 by obstructing linear IgE epitopes.

A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies.

Peanut (Arachis hypogaea) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10 µg/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

Seed-specific overexpression of AtFAX1 increases seed oil content in Arabidopsis.

Biosynthesis of plant seed oil is accomplished through the coordinate action of multiple enzymes in multiple subcellular compartments. Fatty acid (FA) has to be transported from plastid to endoplasmic reticulum (ER) for TAG synthesis. However, the role of plastid FA transportation during seed oil accumulation has not been evaluated. AtFAX1 (Arabidopsis fatty acid export1) mediated the FA export from plastid. In this study, we overexpressed AtFAX1 under the control of a seed specific promoter in Arabidopsis. The resultant overexpression lines (OEs) produced seeds which contained 21-33% more oil and 24-30% more protein per seed than those of the wild type (WT). The increased oil content was probably because of the enhanced FA and TAG synthetic activity. The seed size and weight were both increased accordingly. In addition, the seed number per silique and silique number per plant had no changes in transgenic plants. Taken together, our results demonstrated that seed specific overexpression of AtFAX1 could promote oil accumulation in Arabidopsis seeds and manipulating FA transportation is a feasible strategy for increasing the seed oil content.

2S protein Ara h 7.0201 has unique epitopes compared to other Ara h 7 isoforms and is comparable to 2S proteins Ara h 2 and 6 in basophil degranulation capacity.

BACKGROUND: Screening for specific IgE against 2S albumin proteins Ara h 2 and 6 has good positive predictive value in diagnosing peanut allergy. From the third 2S member Ara h 7, 3 isoforms have been identified. Their allergenicity has not been elucidated. OBJECTIVE: This study investigated the allergenicity of Ara h 7 isoforms compared to Ara h 2 and 6. METHODS: Sensitization of 15 DBPCFC-confirmed peanut-allergic patients to recombinant Ara h 2.0201, Ara h 6.01 and isoforms of recombinant Ara h 7 was determined by IgE immunoblotting strips. A basophil activation test (BAT) was performed in 9 patients to determine IgE-cross-linking capacities of the allergens. Sensitivity to the allergens was tested in 5 patients who were sensitized to at least 1 Ara h 7 isoform, by a concentration range in the BAT. 3D prediction models and sequence alignments were used to visualize differences between isoforms and to predict allergenic epitope regions. RESULTS: Sensitization to Ara h 7.0201 was most frequent (80%) and showed to be equally potent as Ara h 2.0201 and 6.01 in inducing basophil degranulation. Sensitization to Ara h 7.0201 together with Ara h 2.0201 and/or 6.01 was observed, indicating the presence of unique epitopes compared to the other 2 isoforms. Differences between the 3 Ara h 7 isoforms were observed in C-terminal cysteine residues, pepsin and trypsin cleavage sites and 3 single amino acid substitutions. CONCLUSION & CLINICAL RELEVANCE: The majority of peanut-allergic patients are sensitized to isoform Ara h 7.0201, which is functionally as active as Ara h 2.0201 and 6.01. Unique epitopes are most likely located in the C-terminus or an allergenic loop region which is a known allergenic epitope region for Ara h 2.0201 and 6.01. Due to its unique epitopes and allergenicity, it is an interesting candidate to improve the diagnostic accuracy for peanut allergy.