A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ = 0.8 ng ml(-1) in extracts, corresponding to 0.02 µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25 mg g(-1), while the skins contained considerably less, 0.4 mg g(-1). Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5 µg g(-1), or 0.5 ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02 µg g(-1) parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15 µg g(-1) of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.

Enhancing the in vitro Fe(2+) bio-accessibility using ascorbate and cold-set whey protein gel particles.

This paper investigates the possibility for iron fortification of food using a new preparation method for protein gel particles in which iron is entrapped in the presence of ascorbate using cold-set gelation. The effect of ascorbate on the iron-induced cold-set gelation process of whey protein was studied in order to optimize the ratio of iron/ascorbate. Subsequently, the effect of ascorbate on iron bio-accessibility was assessed in vitro. Rheology was used to study the protein gel formation, and the stability of the gel particles was determined by measuring the iron and protein content at different pH. In vitro studies were performed with the TNO Intestinal Model (TIM). Ascorbate appeared to affect the gel formation process and increased the gel strength of the iron-induced cold-set gels at specific iron/ascorbate ratio. With the Fe-protein gel particles being stable at a broad pH range, the release of iron from the particles was studied as a function of time. The low release of iron indicated a good encapsulation efficiency and the capability of whey protein to keep iron bound at different conditions (pH and presence of calcium). Results obtained with the TIM showed that ascorbate, when added to the protein gel particles, was very successful in enhancing the recovery and absorption of iron. The in vitro Fe(2+) bio-accessibility in the presence of ascorbate in iron-protein particles increased from 10% to almost 80%. This suggests that the concept of using protein particles with iron and ascorbate can effectively be used to fortify food products with iron for human consumption.

The effect of the food matrix on in vivo immune responses to purified peanut allergens.

There is little knowledge about the factors that determine the allergenicity of food proteins. One aspect that remains to be elucidated is the effect of the food matrix on immune responses to food proteins. To study the intrinsic immunogenicity of allergens and the influence of the food matrix, purified peanut allergens (Ara h 1, Ara h 2, Ara h 3, or Ara h 6) and a whole peanut extract (PE) were tested in the popliteal lymph node assay (PLNA) and in an oral model of peanut hypersensitivity. In the PLNA, peanut proteins were injected into the hind footpad of BALB/c mice; in the oral exposure experiments C3H/HeOuJ mice were gavaged weekly with PE or allergens in the presence of cholera toxin (CT). Upon footpad injection, none of the allergens induced significant immune activation. In contrast, PE induced an increase in cell number, cytokine production, and activation of antigen-presenting cells. Furthermore, the presence of a food matrix enhanced the immune response to the individual allergens. Oral exposure to the purified allergens in the presence of CT induced specific IgE responses, irrespective of the presence of a food matrix. These results suggest that purified peanut allergens possess little intrinsic immune-stimulating capacity in contrast to a whole PE. Moreover, the data indicate that the food matrix can influence responses to individual proteins and, therefore, the food matrix must be taken into account when developing models for allergenic potential assessment.

Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2.

BACKGROUND: IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of our peanut allergic population. OBJECTIVE: To characterize the novel allergen in order to determine whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h 1-7). METHODS: An IgE-binding protein of <15 kDa was purified and identified via N-terminal sequencing. Its IgE-binding properties were investigated using immunoblotting, basophil degranulation, and skin prick testing. Possible cross-reacting epitopes with other peanut allergens were studied using IgE-immunoblotting inhibition. RESULTS: The purified protein is a monomeric protein with a molecular weight of 14,981 Da as determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h 6, indicating that the allergen is Ara h 6. It is recognized by 20 out of 29 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the degranulation of basophils, even at concentrations below 10 pg/mL, and by positive skin prick reactions. Ara h 6 has homology to Ara h 2, especially in the middle part and at the C-terminal part of the protein. Almost complete inhibition of IgE-Ara h 6 interaction with Ara h 2 demonstrates that at least part of the epitopes of Ara h 6 are cross-reactive with epitopes on Ara h 2. CONCLUSIONS: Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our peanut-allergic patient population and can be considered a clinically relevant peanut allergen.