Kiwifruit Allergen Research Articles

Structural and bioinformatic analysis of the kiwifruit allergen Act d 11, a member of the family of ripening-related proteins

The allergen Act d 11, also known as kirola, is a 17kDa protein expressed in large amounts in ripe green and yellow-fleshed kiwifruit. Ten percent of all kiwifruit-allergic individuals produce IgE specific for the protein. Using X-ray crystallography, we determined the first three-dimensional structures of Act d 11, produced from both recombinant expression in Escherichia coli and from the natural source (kiwifruit). While Act d 11 is immunologically correlated with the birch pollen allergen Bet v 1 and other members of the pathogenesis-related protein family 10 (PR-10), it has low sequence similarity to PR-10 proteins. By sequence Act d 11 appears instead to belong to the major latex/ripening-related (MLP/RRP) family, but analysis of the crystal structures shows that Act d 11 has a fold very similar to that of Bet v 1 and other PR-10 related allergens regardless of the low sequence identity. The structures of both the natural and recombinant protein include an unidentified ligand, which is relatively small (about 250Da by mass spectrometry experiments) and most likely contains an aromatic ring. The ligand-binding cavity in Act d 11 is also significantly smaller than those in PR-10 proteins. The binding of the ligand, which we were not able to unambiguously identify, results in conformational changes in the protein that may have physiological and immunological implications. Interestingly, the residue corresponding to Glu45 in Bet v 1 (Glu46), which is important for IgE binding to the birch pollen allergen, is conserved in Act d 11, even though it is not in other allergens with significantly higher sequence identity to Bet v 1. We suggest that the so-called Gly-rich loop (or P-loop), which is conserved in all PR-10 allergens, may be responsible for IgE cross-reactivity between Bet v 1 and Act d 11.

Characterization of the allergenic potential of proteins: an assessment of the kiwifruit allergen actinidin

Assessment of the potential allergenicity (IgE-inducing properties) of novel proteins is an important challenge in the overall safety assessment of foods. Resistance to digestion with pepsin is commonly measured to characterize allergenicity, although the association is not absolute. We have previously shown that specific IgE antibody production induced by systemic [intraperitoneal (i.p.)] exposure of BALB/c strain mice to a range of proteins correlates with allergenic potential for known allergens. The purpose of the present study was to explore further the utility of these approaches using the food allergen, actinidin. Recently, kiwifruit has become an important allergenic foodstuff, coincident with its increased consumption, particularly as a weaning food. The ability of the kiwifruit allergen actinidin to stimulate antibody responses has been compared with the reference allergen ovalbumin, and with the non-allergen bovine haemoglobin. Haemoglobin was rapidly digested by pepsin whereas actinidin was resistant unless subjected to prior chemical reduction (reflecting intracellular digestion conditions). Haemoglobin stimulated detectable IgG antibody production at relatively high doses (10%), but failed to provoke detectable IgE. In contrast, actinidin was both immunogenic and allergenic at relatively low doses (0.25% to 1%). Vigorous IgG and IgG1 antibody and high titre IgE antibody responses were recorded, similar to those provoked by ovalbumin. Thus, actinidin displays a marked ability to provoke IgE, consistent with allergenic potential. These data provide further encouragement that in tandem with analysis of pepsin stability, the induction of IgE after systemic exposure of BALB/c strain mice provides a useful approach for the prospective identification of protein allergens.

Prevalence and peculiarities of IgE reactivity to kiwifruit pectin methylesterase and its inhibitor, Act d 7 and Act d 6, in subjects allergic to kiwifruit

Kiwifruit is an important cause of allergy. Although several allergens have been identified in this food (www.allergome.org), data on the number and clinical relevance of kiwifruit allergenic proteins are still fragmentary. With the aim of providing a contribution to the description of the complete kiwifruit allergome, we have investigated the immunological features of pectin methylesterase (PME) and its inhibitor (PMEI) purified from the natural source. Specific IgE to PME (Act d 7) and PMEI (Act d 6) were recorded in patients with kiwifruit allergies by immunoblotting, multiplex microarray-based immunoassay and skin testing (ST). Compared to the results obtained by the microarray system, the percentage of subjects detected positive to Act d 6 was much higher in the immunoblotting. Conversely, a similar percentage of subjects positive to Act d 7 was estimated in both the in vitro tests. Few subjects compared to in vitro testing gave a positive response on ST. In the case of Act d 7 this could be ascribed to the glycan side chain recognized by cross-reactive carbohydrate determinants (CCD)-specific IgE. In fact, Act d 7 fell in the cluster of glycoallergens (including Api m 1, Cup a 1, Hor v 17, Ole e 1, Pla a 2) immobilized on the microarray and recognized by CCD-specific IgE. A comparative analysis of different kiwifruit allergens (Act d 1, 2, 5, 6, 7, 11) revealed that only Act d 7 fell in this cluster, whereas Act d 2, although reported to be glycosylated, fell outside, thus suggesting the lack of sugars bound to this molecule. The subsequent biochemical characterization confirmed that Act d 2 is not a glycoallergen. In conclusion, Act d 7 and 6 seem to be rarely involved in causing symptoms in kiwifruit allergic patients. Nevertheless they are useful to understand a single patient profile and the mechanism of protein allergenicity. In addition, in this study the use of a multiplex test system has provided more information than expected, thus contributing to both the immunological and biochemical characterization of the immobilized allergens.

Kiwifruit as a food allergen source

Since its first appearance on the market kiwifruit has become very popular in the human diet due to its pleasant taste, low caloric value and high content of vitamin C. However, kiwifruit allergy has become a frequent cause of type I hypersensitivity in the western society. Molecular basis for kiwifruit allergy has been ascribed to up-to-now 11 identified IgE reactive molecules. They are proteins and glycoproteins with a molecular mass between 50 and 10 kDa. The major kiwifruit allergen is a cysteine protease denoted as Act d 1, which represents 50% of the soluble protein extract. Due to a difference in the abundance of protein components and biological activity, the quality of kiwifruit extracts intended for allergy diagnosis can vary in content and amount of IgE reactive molecules. In addition, the quality of allergen extracts for allergy diagnosis depends on the fruit ripening stage and storage conditions. In terms of clinical reactivity it has become evident that kiwifruit allergy is not a homogeneous disorder. Different patterns of IgE reactivity accompany several clinical subgroups that have been identified in different geographical regions. In the last decade an enormous progress has been made in isolation and characterization of kiwifruit allergens. This paper will give an overview of the structural features of kiwifruit allergens.

Comparative transcript profiling by SuperSAGE identifies novel candidate genes for controlling potato quantitative resistance to late blight not compromised by late maturity.

Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR.

Kiwifruit allergy across Europe: Clinical manifestation and IgE recognition patterns to kiwifruit allergens

Kiwifruit is a common cause of food allergy. Symptoms range from mild to anaphylactic reactions. We sought to elucidate geographic differences across Europe regarding clinical patterns and sensitization to kiwifruit allergens. Factors associated with the severity of kiwifruit allergy were identified, and the diagnostic performance of specific kiwifruit allergens was investigated. This study was part of EuroPrevall, a multicenter European study investigating several aspects of food allergy. Three hundred eleven patients with kiwifruit allergy from 12 countries representing 4 climatic regions were included. Specific IgE to 6 allergens (Act d 1, Act d 2, Act d 5, Act d 8, Act d 9, and Act d 10) and kiwifruit extract were tested by using ImmunoCAP. Patients from Iceland were mainly sensitized to Act d 1 (32%), those from western/central and eastern Europe were mainly sensitized to Act d 8 (pathogenesis-related class 10 protein, 58% and 44%, respectively), and those from southern Europe were mainly sensitized to Act d 9 (profilin, 31%) and Act d 10 (nonspecific lipid transfer protein, 22%). Sensitization to Act d 1 and living in Iceland were independently and significantly associated with severe kiwifruit allergy (odds ratio, 3.98 [P = .003] and 5.60 [P < .001], respectively). Using a panel of 6 kiwifruit allergens in ImmunoCAP increased the diagnostic sensitivity to 65% compared with 20% for skin prick tests and 46% ImmunoCAP using kiwi extract. Kiwifruit allergen sensitization patterns differ across Europe. The use of specific kiwifruit allergens improved the diagnostic performance compared with kiwifruit extract. Sensitization to Act d 1 and living in Iceland are strong risk factors for severe kiwifruit allergy.

Actinidin, a protease from kiwifruit, induces changes in morphology and adhesion of T84 intestinal epithelial cells

Actinidin belongs to the papain-like family of cysteine proteases and is a major kiwifruit allergen. In this study, the effect of actinidin on cellular morphology and adhesion of T84 intestinal cells was investigated. Both rounding and detachment of T84 cells were observed upon actinidin treatment. The morphological changes and cell desquamation was protease-dependent, as well as time- and concentration-dependent. Changes of intercellular adhesion and adhesion of epithelial cells to collagen upon actinidin treatment could be responsible for the cell rounding and give rise to discontinuous breaches in the epithelial monolayer observed in this study. Actinidin’s action on cell morphology, adhesion and monolayer integrity were not due to compromised viability of T84 epithelial cells, as confirmed by MTT assay and flow cytometric analysis of the cell cycle. Damage to the epithelial monolayer of the intestine induced by actinidin should be further evaluated as an important factor in the development of kiwifruit allergy and other intestinal disorders.

Sensitization profiles to purified plant food allergens among pediatric patients with allergy to banana

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize β-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana β-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana β-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency.

The performance of a component-based allergen microarray for the diagnosis of kiwifruit allergy

Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests.

In Vivo Digestion of a Thaumatin-Like Kiwifruit Protein in Rats

Food allergens must exhibit sufficient gastrointestinal stability to reach the intestinal mucosa where absorption and sensitization can occur. Therefore, investigation of protein stability within the gastrointestinal tract may provide a prospective test for the allergenic potential of novel proteins. The aim of this work was to examine the effect of the fruit matrix and purified pectin on the digestion in vivo of kiwifruit allergens in the rat gastrointestinal system. The major kiwi allergen, Act d 2, was quantified in several compartments of the gastrointestinal tract by a monoclonal antibody-based ELISA. Protein intactness was demonstrated by immunoblot. Under conditions of complex food digestion in vivo, a pepsin-labile protein survived passage from the stomach to the caecum during a 3-h period. Decay of Act d 2 in the rat gut exhibited an exponential pattern. Ingestion of kiwifruit was followed by a decrease in both total and specific pepsin activity. When purified, Act d 2 allergen was consumed together with pure apple pectin; both gastric acidity as well as specific and total pepsin activity declined and thus protected 23% of the ingested allergen from digestion for 90 min. In conclusion, ingestion of pectin-rich fruits and particularly pectin supplements may have a protective action on pepsin-labile allergens and prolong their survival in both gastric and duodenal juices, enabling efficient uptake and presentation to the immune system.

Component-resolved diagnosis of kiwifruit allergy with purified natural and recombinant kiwifruit allergens

Kiwifruit is one of the most common causes of food allergic reactions. Component-resolved diagnostics may enable significantly improved detection of sensitization to kiwifruit. To evaluate the use of individual allergens for component-resolved in vitro diagnosis of kiwifruit allergy. Thirty patients with a positive double-blind placebo-controlled food challenge to kiwifruit, 10 atopic subjects with negative open provocation to kiwifruit, and 5 nonatopic subjects were enrolled in the study. Specific IgE to 7 individual allergens (nAct d 1-5 and rAct d 8-9) and allergen extracts was measured by ImmunoCAP. The diagnostic sensitivities of the commercial extract and of the sum of single allergens were 17% and 77%, respectively, whereas diagnostic specificities were 100% and 30%. A combination of the kiwi allergens Act d 1, Act d 2, Act d 4, and Act d 5 gave a diagnostic sensitivity of 40%, whereas diagnostic specificity remained high (90%). Exclusion of the Bet v 1 homolog recombinant (r) Act d 8 and profilin rAct d 9 from this allergen panel reduced sensitivity to 50% but increased specificity to 40%. Kiwifruit-monosensitized patients reacted more frequently (P < .001) with Act d 1 than polysensitized patients, whereas the latter group reacted more frequently with rAct d 8 (P = .004). Use of single kiwifruit allergen ImmunoCAP increases the quantitative test performance and diagnostic sensitivity compared with the commercial extract. Bet v 1 homolog and profilin are important allergens in pollen-related kiwifruit allergy, whereas actinidin is important in monoallergy to kiwifruit, in which symptoms are often more severe.

Characterization of Bet v 1-related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin‐like protein

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

Cover Picture – Mol. Nutr. Food Res. 10/2008

AbstractRegular issues provide a wide range of research and review articles covering all aspects of Molecular Nutrition &amp; Food Research.Selected topics of issue 10 are:Urinary excretion of strawberry anthocyanins .Isoflavone‐Poor Soy Protein &amp; Reduced mammary tumor progression Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Mediterranean diet and cardioprotection Meat‐based functional foods for dietary equilibrium omega‐6/omega‐3 Modulation of Endothelial Cell Gene Expression by Green Tea

Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens

Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

The effect of digestion and pH on the allergenicity of kiwifruit proteins

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.

Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms

Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients. To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population. A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs. The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity. Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

Quantification of the thaumatin‐like kiwi allergen by a monoclonal antibody‐based ELISA

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.

Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom

Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy. To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera. Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA. A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

A matrix effect in pectin‐rich fruits hampers digestion of allergen by pepsin in vivo and in vitro

It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.