High frequency of IgE sensitization towards kiwi seed storage proteins among peanut allergic individuals also reporting allergy to kiwi.

BACKGROUND: IgE sensitization to storage proteins from nuts and seed is often related to severe allergic symptoms. There is a risk of immunological IgE cross-reactivity between storage proteins from different species. The potential clinical implication of such cross-reactivity is that allergens other than the known sensitizer can cause allergic symptoms. Previous studies have suggested that kiwi seed storage proteins may constitute hidden food allergens causing cross-reactive IgE-binding with peanut and other tree nut homologs, thereby mediating a potential risk of causing allergy symptoms among peanut ant tree nut allergic individuals. The objective of this study was to investigate the degree of sensitization towards kiwi fruit seed storage proteins in a cohort of peanut allergic individuals. METHODS: A cohort of 59 adolescents and adults with peanut allergy was studied, and self reported allergies to a number of additional foods were collected. Quantitative IgE measurements to seed storage proteins from kiwi and peanut were performed. RESULTS: In the cohort, 23 out of the 59 individuals were reporting kiwi fruit allergy (39%). The frequency of IgE sensitization to kiwi fruit and to any kiwi seed storage protein was higher among peanut allergic individuals also reporting kiwi fruit allergy (P = 0.0001 and P = 0.01). A positive relationship was found between IgE levels to 11S globulin (r = 0.65) and 7S globulin (r = 0.48) allergens from kiwi and peanut, but IgE levels to 2S albumin homologs did not correlate. Patients reporting kiwi fruit allergy also reported allergy to hazelnut (P = 0.015), soy (P < 0.0001), pea (P = 0.0002) and almond (P = 0.016) to a higher extent than peanut allergic individuals without kiwi allergy. CONCLUSIONS: Thirty-nine percent of the peanut allergic patients in this cohort also reported kiwi fruit allergy, they displayed a higher degree of sensitization to kiwi storage proteins from both kiwi and peanut, and they also reported a higher extent of allergy to other nuts and legumes. On the molecular level, there was a correlation between IgE levels to 11S and 7S storage proteins from kiwi and peanut. Taken together, reported symptoms and serological findings to kiwi in this cohort of patients with concurrent allergy to peanut and kiwi fruit, could be explained by a combination of cross-reactivity between the 11S and 7S globulins and co-sensitization to the 2S albumin Act d 13.

Affinity purification of egg-white allergens for improved component-resolved diagnostics.

BACKGROUND: Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD. METHODS: Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals. RESULTS: Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%). CONCLUSIONS: In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.

Expression, purification, cross-reactivity and homology modeling of peanut profilin.

Plant profilins are known pan-allergens involved in the cross-reactions between pollen and plant foods. Peanut profilin, Ara h 5, is one of the important peanut allergens. Presently, most immunological, biochemical and structural studies on peanut allergens have focused on the three major allergens (Ara h 1, 2 and 3). Here Ara h 5 was cloned, expressed in Escherichia coli, Rosetta2(DE3) (Novagen), purified using a combination of ammonium sulfate fractionation and size-exclusion chromatography and yielded a total of 29 mg/l of culture. IgE reactivity was assayed using multiplexed microarray with other peanut allergens (Ara h 1, 2, 3, and 8) and birch (Bet v 2) and timothy (Phl p 2) profilin using sera from peanut allergic Swedish patients. Using homology modeling, Ara h 5 structure was also generated, compared against other profilins and utilized to predict surface-exposed residues potentially forming epitopes. The allergen was recognized by 3 out of 33 sera (9.1%). IgE reactivity to Ara h 5 also coincided with that of two other profilins, Phl p 12 and Bet v 2, confirming cross-reactivity. Interestingly, IgE reactivity to Ara h 5 was higher than above-mentioned profilins which may be indicating specificity of sera towards peanut profilin. Eight surface-exposed epitopes were predicted and verified against experimentally validated sequential epitopes. Three epitopes (#1, 5 and 7) mostly located at the accessible loops and neutral to relatively electropositive sites were found common among profilins, which should be involved in cross-reactivity. A specific putative epitope (#4) was also found which may explain the relative high IgE reactivity to Ara h 5 as compared to the other profilins. Due to its close relation to other allergenic profilins, Ara h 5 could be used as a model and allergen of choice for profilin allergy diagnosis.

Purification and characterization of the major oak pollen allergen Que a 1 for component-resolved diagnostics using ImmunoCAP.

BACKGROUND: The aim of this study was to purify the major oak pollen allergen, Que a 1, to perform biochemical and immunological characterization of the allergen and to develop an experimental native (n) Que a 1 ImmunoCAP(R). METHODS: Que a 1 was purified from oak pollen extract using affinity chromatography and characterized by SDS-PAGE, two-dimensional (2D) PAGE, mass spectrometry (MS), N-terminal sequencing and specific IgE inhibition on ImmunoCAP. Samples from 16 subjects sensitized to oak pollen were analyzed by ImmunoCAP for IgE reactivity to nQue a 1, and recombinant (r)Bet v 1 and 2 (profilin). They were also studied in IgE immunoblotting. RESULTS: The purity of nQue a 1 was >95%, since a single band was observed on silver-stained SDS-PAGE. The identity was verified by MS analysis, and 2D-PAGE revealed several isoforms. The obtained N-terminal sequence of 50-amino-acid residues from nQue a 1 showed a 58-74% sequence identity with other pathogenesis-related class 10 allergens. Specific IgE inhibition verified a preserved immunoreactivity (70-92% inhibition). All subjects were sensitized to Que a 1 and Bet v 1, and two to profilin. The IgE antibody levels to nQue a 1 were generally lower than to rBet v 1. The obtained results correlated well with IgE immunoblotting. CONCLUSIONS: We present a highly purified and extensively characterized preparation of nQue a 1. Que a 1 seems to be an allergen of equal importance in oak pollen as Bet v 1 in birch pollen. An nQue a 1 ImmunoCAP will be useful in component-resolved diagnostics.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.