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Pru p 7 (also named Peamaclein) is a member of the gibberellin-regulated protein family, which is the latest foodborne allergenic protein identified in peach. In this paper, the prokaryotic expression and identification of Pru p 7 were performed, and the protein properties, structure, and homology were analyzed. In addition, a preliminary screening of B-cell linear epitopes of Pru p 7 was performed by the bioinformatics software prediction method, and three epitopes were identified using slot-blot immune microarray assay combined with an immune score matrix (P-1, AA16-21, AGYQER; P-2, AA40-46, TYGNKDE; P-3, AA52-59, DLKNSKGN). Moreover, the electrostatic potential of these epitopes was analyzed, and the stability after ultrahigh pressure treatment was also verified. Finally, the amino acids that play key immune roles in the epitopes were obtained by amino acid mutations. These results may contribute to the further understanding of Pru p 7 and the prevention of peach allergy.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans- acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1 , and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1 -gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T 1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I ( 151 SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG 181 ) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 ( 157 DQMPRRFYLANGNQE 170 ) showed allergenicity, and after replacing it with alanine, it was found that amino acids D 157 , Q 158 , M 159 , and Y 164 were the key amino acids that affected its antigenicity, while Q 158 , M 159 , R 162 , and N 168 affected allergenicity.

Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients. These reactions can be due to the patient's previous sensitization to the major birch pollen allergen Bet v 1, by which IgE generated in response to Bet v 1 cross-reacts with the structurally related strawberry Fra a 1 protein family. Fra a 1.02 is the most expressed paralog in ripe strawberries and is highly allergenic. To better understand the molecular mechanisms regulating this allergic response, we have determined the three-dimensional structure of Fra a 1.02 and four site-directed mutants that were designed based on their positions in potential epitopes. Fra a 1.02 and mutants conform to the START fold. We show that the cross-reactivity of all the mutant variants to IgE from patients allergic to Bet v 1 was significantly reduced without altering the conserved structural fold, so that they could potentially be used as hypoallergenic Fra a 1 variants for the generation of vaccines against strawberry allergy in atopic patients.

Hordeins are the major barley seed storage proteins and are elicitors of celiac disease. Attempts to reduce the hordein level in barley have been made; however, the resultant pleiotropic effects are less understood. Here, data-independent acquisition mass spectrometry was used to measure proteome-wide abundance differences between wild-type and single hordein-null barley lines. Using comparative quantitative proteomics, we detected proteome-wide changes (∼59%) as a result of the specific reduction in hordein proteins. The comparative analysis and functional annotation revealed an increase in non-gluten storage proteins, such as globulins and lipid transfer proteins, and proteins rich in essential amino acids in the null lines. This study yields an informative molecular portrait of the hordein-null lines and the underlying mechanisms of storage protein biosynthesis. This study indicates the extent to which protein content can be manipulated without biological consequence, and we envision its wide-scale application for studying modified crops.

Human noroviruses (HuNoVs) are among the main pathogens causing acute nonbacterial gastroenteritis. Histo-blood group antigens (HBGAs) are widely accepted receptors for HuNoV specific binding. HBGA-like substances in produce are also considered as the critical ligands for capture of HuNoVs. However, the composition of viral ligands from food substrates remains unknown. In this study, an oligosaccharide (H2N2F2) was captured and isolated from romaine lettuce extract by a bacterial surface display system. Using electrospray ionization mass spectrometry and tandem mass spectrometry, it was shown that H2N2F2 was most likely to be a chimera of type A, H, and Lewis a HBGAs. The composition was consistent with our ELISA results using a panel of monoclonal antibodies against HBGAs. Our results revealed a possible interaction mechanism between HuNoVs and romaine lettuce. Better understanding of the interaction of HuNoVs with easily contaminated produce will ultimately aid in the control of and reduction in disease outbreaks.

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.

Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X] 3 C-[X] 10-12 -C[X] 3 C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.

Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.

Freshly consumed peaches ( Prunus persica L. Batsch) can cause allergic reactions in the worldwide population because of the presence of four classes of allergens (Pru p 1, Pru p 2, Pru p 3, and Pru p 4). Fruit bagging has been widely practiced in peach cultivation to improve fruit quality; however, its effect on the expression of peach allergen-encoding genes remains unknown. In this study, the influence of fruit bagging with opaque paper bags on the major peach allergen-encoding genes, including Pru p 1.01, Pru p 1.06B, Pru p 2.01B, Pru p 2.02, Pru p 3.01, Pru p 4.01, and Pru p 4.02, were measured by means of real-time PCR. A significant reduction in transcript accumulation was observed for all of the selected genes in the epicarps of the bagged peach fruits, whereas slight increases were observed in the mesocarps for these genes, with the two exceptions of Pru p 2.02 and Pru p 3.01. For most of these genes, much higher transcripts were determined in the epicarps than in the mesocarps. Taken together, a significant reduction in the transcription rate of the allergen-encoding genes in the whole peach fruit was achieved by shading with opaque paper bags. According to these data, modifications in growing practices of peach may help to obtain fruits with lower levels of allergens and thus contribute to reducing potential allergenic risks in consumers.

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

The consumption of pomegranate is increasing as it is considered a health-promoting food. Nevertheless, it can trigger allergic reactions, sometimes severe. The LTP Pun g 1 is the only pomegranate allergen so far reported. Based on preliminary clinical observations, the main aim of this study was the investigation of still unknown allergens contained in this fruit. Pommaclein, a homologue of peamaclein, the peach allergen Pru p 7, was isolated, identified by protein sequencing, and characterized as an IgE-binding protein by different test systems. RP-HPLC protein profiles revealed significant variations of LTP and pommaclein content in the red pulp of selected cultivars and accessions. Conversely, the mesocarp appeared free of proteins and much richer in antioxidants. In conclusion, a new allergen has been identified, and it could contribute to improving allergy diagnosis. The study highlights that pomegranate mesocarp could represent a rich and safe source of nutraceuticals also for allergic subjects.

Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

Tree nuts as a group cause a significant number of fatal anaphylactic reactions to foods. Walnuts (Juglans spp.) are one of the leading causes of allergic reactions to tree nuts in the U.S. and Japan. The purpose of this study was to purify and characterize potential food allergens from black walnut. Here, we report the isolation of the black walnuts allergen Jug n 4 (an 11S globulin) by ammonium sulfate precipitation, hydrophobic interaction, and size exclusion chromatography. Reducing SDS-PAGE analysis indicated that purified Jug n 4 consists of three major bands. N-Terminal sequencing data of these bands indicated that they were the results of a post-transcriptional protease cleavage of the mature protein at a site that consists of a known conserved protease recognition motif, NGXEET. Western blot experiments revealed that 32% of the sera from 25 patients with double-blind, placebo-controlled clinical walnut allergy contained IgE antibodies that recognized Jug n 4, indicating that it is a walnut allergen. Identifying this and additional allergens may facilitate the understanding of the allergenicity of seed storage proteins in tree nuts and their cross-reactivity.

The strawberry fruit proteins Fra a 1.01E-1.08 are homologues of the major birch pollen allergen Bet v 1. Three of the proteins are known to have essential biological functions in pigment formation during fruit ripening and seem to be responsible for allergic reactions to strawberry fruit. We evaluated the cross-reactive allergenic potential of these putative strawberry allergens in patients allergic to birch pollen. Activation of basophils of eight atopic patients was studied using different concentrations of Fra a 1 isoforms. Bet v 1a was used as control and as atopic patient selection criterion. Although Fra a 1.01E-1.08 have amino acid sequence identities of 74.5-97.5% with Fra a 1.02, the basophil activation mediated by the eight Fra a 1 proteins differed substantially. Fra a 1.03 and Fra a 1.02 showed the highest activation of basophils, 73 and 66% of total basophils, respectively. On the basis of the high relative expression of the gene Fra a 1.02 in ripe strawberry fruits of allergenic varieties, Fra a 1.02 was identified as the main strawberry allergen of the Bet v 1 superfamily. Knowledge of the allergenic potential of Fra a 1.02/1.03 will help to improve food safety and can serve as a valuable marker for the development of red-fruited hypoallergenic strawberry cultivars.

Compared to β-conglycinin, glycinin contains 3-4 times the methionine and cysteine (sulfur-containing amino acids), accounting for approximately 40 and 30%, respectively, of the total storage protein in soybean. Increasing the soybean storage protein content while improving the ratio of glycinin to β-conglycinin is of great significance for soybean breeding and soy food products. The objective of this study is to analyze the genetic mechanism regulating the glycinin and β-conglycinin contents of soybean by using a recombinant inbred line (RIL) population derived from a cross between Kefeng No. 1 and Nannong 1138-2. Two hundred and twenty-one markers were used to map quantitative trait loci (QTLs) for glycinin (11S) and β-conglycinin (7S) contents, the ratio of glycinin to β-conglycinin (RGC), and the sum of glycinin and β-conglycinin (SGC). A total of 35 QTLs, 3 pairs of epistatic QTLs, and 5 major regions encompassing multiple QTLs were detected. Genes encoding the subunits of β-conglycinin were localized to marker intervals sat_418-satt650 and sat_196-sat_303, which are linked to RGC and SGC; marker sat_318, associated with 11S, 7S, and SGC, was located near Glyma10g04280 (Gy4), which encodes a subunit of glycinin. These results, which take epistatic interactions into account, will improve our understanding of the genetic basis of 11S and 7S contents and will lay a foundation for marker-assisted selection (MAS) breeding of soybean and improving the quality of soybean products.

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

In this study a fast and simple purification procedure for the three known allergens from cashew (7S globulin Ana o 1, 11S globulin Ana o 2, and 2S albumin Ana o 3) is described. The purified allergens are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein stain, and protein identification. The purified proteins still bind IgE, and this IgE binding varied between different pools of patient serum. Ana o 1 was found to be a glycoprotein. Ana o 3 has been studied more in detail to identify both the small and large subunits, both displaying microheterogeneity, and epitope mapping of Ana o 3 has been performed.

The ω5-gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which ω5-gliadin genes were silenced by RNA interference. Sera from 7 of 11 WDEIA patients showed greatly reduced levels of immunoglobulin E (IgE) reactivity to ω5-gliadins in both transgenic lines. However, these sera also showed low levels of reactivity to other gluten proteins. Sera from three patients showed the greatest reactivity to proteins other than ω5-gliadins, either high-molecular-weight glutenin subunits (HMW-GSs), α-gliadins, or non-gluten proteins. The complexity of immunological responses among these patients suggests that flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. However, the introduction of wheat lacking ω5-gliadins could reduce the number of people sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy.

Wheat [Triticum aestivum (T.a.)] ingestion can cause a specific allergic reaction, which is called wheat-dependent exercise-induced anaphylaxis (WDEIA). The major allergen involved is ω-5 gliadin, a gluten protein coded by genes located on the B genome. Our aim was to study the immunoreactivity of proteins in Triticum monococcum (einkorn, T.m.), a diploid ancestral wheat lacking B chromosomes, for possible use in the production of hypoallergenic foods. A total of 14 patients with a clear history of WDEIA and specific immunoglobulin E (IgE) to ω-5 gliadin were enrolled. Skin prick test (SPT) with a commercial wheat extract and an in-house T.a. gluten diagnostic solution tested positive for 43 and 100% of the cases, respectively. No reactivity in patients tested with solutions prepared from four T.m. accessions was observed. The immunoblotting of T.m. gluten proteins performed with the sera of patients showed different IgE-binding profiles with respect to T.a., confirming the absence of ω-5 gliadin. A general lower immunoreactivity of T.m. gluten proteins with scarce cross-reactivity to ω-5 gliadin epitopes was assessed by an enzyme-linked immunosorbent assay (ELISA). Given the absence of reactivity by SPT and the limited cross-reactivity with ω-5 gliadin, T.m. might represent a potential candidate in the production of hypoallergenic bakery products for patients sensitized to ω-5 gliadin. Further analyses need to be carried out regarding its safety.

Transgenic peanut (Arachis hypogaea L.) containing a gene designed for RNA interference (RNAi) showed stable complete silencing of Ara h 2 and partial silencing of Ara h 6, two potent peanut allergens/proteins, along with minimal collateral changes to other allergens, Ara h 1 and Ara h 3, across three generations (T3, T4, and T5) under field conditions. Different soil sulfur levels (0.012, 0.3, and 3.0 mM) differentially impacted sulfur-rich (Ara h 2, Ara h 3, and Ara h 6) versus sulfur-poor (Ara h 1) proteins in non-transgenic versus transgenic peanut. The sulfur level had no effect on Ara h 1, whereas low sulfur led to a significant reduction of Ara h 3 in transgenic and non-transgenic seeds and Ara h 2 and Ara h 6 in non-transgenic but not in transgenic peanuts because these proteins already were reduced by gene silencing. These results demonstrate stability of transgene expression and the potential utility of RNAi in allergen manipulation.

Biochemical characterizations of food allergens are required for understanding the allergenicity of food allergens. Such studies require a relatively large amount of highly purified allergens. The level of Pru du 4 in almond is low, and its expression in a soluble form in Escherichia coli required an expression tag. An MBP tag was used to enhance its expression and solubility. Sumo was used for the first time as a peptidase recognition site. The expression tag was removed with a sumo protease, and the resulting wild-type Pru du 4 was purified chromatographically. The stability of the allergen was investigated with chemical denaturation. The Gibbs free energy of Pru du 4 folding-unfolding transition was determined to be 5.4 ± 0.7 kcal/mol.

Top-down approaches for quantification of proteins based on separation and mass spectrometric assays hold promise due to their high specificity and avoidance of both proteolytic steps and need for generation of monoclonal antibodies. In this study, a 2DLC-UV/MS assay was developed for the simultaneous quantification of two intact soybean allergens, hydrophobic protein from soybean (HPS) and Gly m 4. Both of these allergens were purified from soybean seeds followed by complete characterization. The method validation consisted of evaluating linearity, precision, and recovery. A linear relationship (R(2) > 0.99) between concentrations of the two proteins and their respective peak areas was observed over the concentration ranges from 6.9 to 355.1 μg/mL and from 11.9 to 599.8 μg/mL for Gly m 4 and HPS, respectively. For the 4 day validation study, precision range (%CV) was observed to be from 4.7 to 9.2% for HPS and from 6.3 to 9.4% for Gly m 4. The assay recovery range (%RE) was observed to be from -1.1 to -13.7% for HPS and from -3.5 to 15.2% for Gly m 4. The assay was applied on 10 non-transgenic commercial lines to quantify the relative levels of the two allergens. The HPS and Gly m 4 levels ranged from 64 to 479 μg/g and from 204 to 637 μg/g, respectively. To the best of the authors' knowledge, this represents the first 2DLC-UV/MS assay for the simultaneous quantitation of selected allergens at the intact level.

The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5 kD band, and the identity of Ara h 6 was confirmed by liquid chromatography-tandem mass spectrometry. Ara h 6 showed a higher seroprevalence in chimeric IgE enzyme-linked immunosorbent assay (n = 54) but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.

Several allergens have been identified and characterized in the genus Citrus, which belongs to the germin-like proteins (GPLs), profilins, and non-specific lipid transfer proteins (nsLTPs). In this work, in silico sequence analysis, protein purification, mass spectrometry identification, and the spectral counting method were integrated to identify new putative allergens of Citrus clementina and their expression level in the fruit peel. The in silico analysis revealed fifteen new sequences belonging to GLPs (Cit cl 1), and two more belonging to nsLTPs (Cit cl 3). No other new sequences were found as regards profilins (Cit cl 2). Each putative allergen from fruit peel was obtained using different protein extraction methods, and the protein sequences of the putative allergens were identified by means of LTQ-Orbitrap XL mass spectrometer. The spectral counting strategy revealed that Cit cl 1 had a higher expression level than Cit cl 2 and Cit cl 3. To predict the quaternary structure and deduced function of Cit cl 1, its primary sequence was used as a template to search a homologous protein structure in the RCSB PDB Database, getting high correspondence with the oxalate oxidase protein in barley.

Rice, the staple food of south and east Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16, 26, 33, and 56 kDa have been identified as allergens. Recently, it was documented that the 56 kDa rice allergen was responsible for rice-induced anaphylaxis. The 14-16 kDa allergens have been identified as α-amylase inhibitors; the 26 kDa protein has been identified as α-globulin; and the 33 kDa protein has been identified as glyoxalase I. However, the identity of the 56 kDa rice allergen has not yet been determined. In this study, we demonstrate that serum from patients allergic to maize shows IgE binding to a 56 kDa protein that was present in both maize and rice but not in the oil seeds soybean and peanut. The 56 kDa IgE-binding protein was abundant in the rice endosperm. We have purified this protein from rice endosperm and demonstrated its reactivity to IgE antibodies from the serum of maize-allergic patients. The purified protein was subjected to matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry analysis, resulting in identification of this rice allergen as granule-bound starch synthase, a product of the Waxy gene. Immunoblot analysis using protein extracts from a waxy mutant of rice revealed the absence of the 56 kDa IgE-binding protein. Our results demonstrate that the 56 kDa rice allergen is granule-bound starch synthase and raise the possibility of using waxy mutants of rice as a potential source of the hypoallergenic diet for patients sensitized to the 56 kDa rice allergen.

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.

Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

The seeds of peas (Pisum sativum) and chickpeas (Cicer arietinum) expressing a gene for α-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are protected from damage by old world bruchids (pea and cowpea weevils). Here, we used electrospray ionization time-of-flight mass spectrometry to compare the post-translational modifications of αAI from transgenic sources with the processed forms of the protein from several bean varieties. All sources showed microheterogeneity with differences in the relative abundance of particular variants due to differences in the frequency of addition of glycans, variable processing of glycans, and differences of C-terminal exopeptidase activity. The structural variation among the transgenics was generally within the range of the bean varieties. Previously, mice showed allergic reactions following ingestion of transgenic pea αAI but not bean αAI. Here, only minor differences were observed following intraperitoneal sensitization. Both of the transgenic pea and bean forms of αAI elicited Th1 and Th2 antibody isotype responses, suggesting that both proteins are immunogenic and could potentially be allergenic.

This paper presents a real-time PCR method allowing the detection of traces of white mustard ( Sinapis alba ) in complex food matrices. The primers and the probe are targeted at the gene coding for S. alba MADS D. The real-time PCR method was found to be specific for white mustard and did not show any cross-reactivity with 67 biological species, including 12 members of the Brassicaceae family. The limit of detection, determined by analyzing serially diluted white mustard DNA extracts, was 1 pg of white mustard DNA/μL, corresponding to 5 pg of white mustard DNA. In model sausages, the limit of detection was found to be 0.001% white mustard (corresponding to 10 ppm or 10 mg/kg). The real-time PCR method was applied to verify the correct declaration of 20 foodstuffs purchased from Austrian supermarkets. White mustard DNA was detected in one of three samples labeled with "may contain traces of mustard" and in one of seven samples without any information on the presence of mustard.

Many tree nuts are considered to be a serious problem in food safety, because of the presence of causative factors in IgE-mediated food allergies. Among these, hazelnut (Corylus avellana L.) seeds are largely used in a range of confectionery products and contain many well-characterized allergens. DNA-based methods and ELISA tests may prove to be useful to assess the presence of hidden ingredients in foods. The aim of this work was the development and validation of a species-specific SYBR Green I real-time PCR protocol for the detection of hazelnut in foods. A novel efficient primer pair on the Cor a 8 genomic coding region was designed by preparing a plasmid vector-based internal reference standard to calibrate the PCR. A good sensitivity, down to 20 (genomic) and 15 (plasmid) DNA copies, was established. All of the commercial samples considered in our study (containing hazelnut as ingredient or as a potential trace cross-contamination) were effectively amplified by PCR, showing a perfect correspondence with an ELISA commercial test, employed as a reference standard method.

The paper describes the development and validation of a duplex real-time PCR method allowing the simultaneous detection of traces of potentially allergenic sesame and hazelnut in food. For the detection of sesame and hazelnut, the genes coding for two major allergenic proteins, Ses i 1 and Cor a 1, were selected. The duplex real-time PCR assay did not show any cross-reactivity with 25 common food ingredients from sesame and/or hazelnut containing foods. Analysis of serially diluted sesame/hazelnut DNA resulted in good linearity up to a dilution of 1:10000 (corresponding to 10 pg microL(-1) or 50 pg). Sesame and hazelnut could be detected in blank whole meal cookies which had been spiked with 0.005% sesame and 0.005% hazelnut. The applicability of the real-time PCR assay for determining sesame and hazelnut in different food matrices was investigated by analyzing 30 commercial foodstuffs comprising salty snacks, cookies, chocolates, creams, mueslis and muesli bars.

The fruits of Rosaceae species may frequently induce allergic reactions in both adults and children, especially in the Mediterranean area. In peach, true allergens and cross-reactive proteins may cause hypersensitive reactions involving a wide diversity of symptoms. Three known classes of allergenic proteins, namely, Pru p 1, Pru p 3, and Pru p 4, have been reported to be mostly involved, but an exhaustive survey of the proteins determining the overall allergenic potential, their biological functions, and the factors affecting the expression of the related genes is still missing. In the present study, the expression profiles of some selected genes encoding peach allergen isoforms were studied during fruit growth and development and upon different fruit load and light radiation regimens. The results indicate that the majority of allergen-encoding genes are expressed at their maximum during the ripening stage, therefore representing a potential risk for peach consumers. Nevertheless, enhancing the light radiation and decreasing the fruit load achieved a reduction of the transcription rate of most genes and a possible decrease of the overall allergenic potential at harvest. According to these data, new growing practices could be set up to obtain hypoallergenic peach fruits and eventually combined with the cultivation of hypoallergenic genotypes to obtain a significant reduction of the allergenic potential.

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.

Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide.

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.