Characterisation of potential novel allergens in the fish parasite Anisakis simplex.

The parasitic nematode Anisakis simplex occurs in fish stocks in temperate seas. A. simplex contamination of fish products is unsavoury and a health concern considering human infection with live larvae (anisakiasis) and allergic reactions to anisakid proteins in seafood. Protein extracts of A. simplex produce complex band patterns in gel electrophoresis and IgE-immunostaining. In the present study potential allergens have been characterised using sera from A. simplex-sensitised patients and proteome data obtained by mass spectrometry. A. simplex proteins were homologous to allergens in other nematodes, insects, and shellfish indicating cross-reactivity. Characteristic marker peptides for relevant A. simplex proteins were described.

Structural and immunological characterization of recombinant Pan b 1, a major allergen of northern shrimp, Pandalus borealis.

BACKGROUND: Shellfish allergy is one of the major causes of life-threatening allergic reactions to food. The shrimp species Pandalus borealis is the commercially most important coldwater shrimp species, and its protein extract is commonly used in shrimp allergy diagnostics. However, the DNA sequence of its major allergen, tropomyosin, designated Pan b 1, was not previously described. Our aim was to identify the cDNA sequence of Pan b 1 and to generate a recombinant protein with similar structure and allergenicity as the natural protein. METHODS: P. borealis shrimps were caught in the Oslofjord (Norway). cDNA from Pan b 1 was generated, an N-terminal histidine tag was added, and the protein was expressed in Escherichia coli. The recombinant Pan b 1 was characterized by structural and IgE-binding studies and investigated further with basophil activation tests (BATs) and skin prick tests (SPTs) on Norwegian shrimp-allergic individuals. RESULTS: The open reading frame encoded 284 amino acids that shared 97-100% identity with other shrimp tropomyosins. Mass spectroscopy of natural Pan b 1 confirmed the protein's molecular mass and indicated the absence of posttranslational modifications. Circular dichroism spectroscopy revealed virtually identical spectra between recombinant and natural Pan b 1, which together with native PAGE and size exclusion chromatography results indicated a similar structure. Furthermore, immunoblot and ELISA studies as well as BATs and SPTs showed equivalent results of recombinant and natural Pan b 1. CONCLUSION: A recombinant tropomyosin from P. borealis was generated that can be used in diagnostics and further studies on tropomyosin allergenicity and specific immunotherapy.

Severe allergic reactions to food in Norway: a ten year survey of cases reported to the food allergy register.

The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two "new" allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies.

Characterization of potential allergens in fenugreek (Trigonella foenum-graecum) using patient sera and MS-based proteomic analysis.

BACKGROUND: Fenugreek is a legume plant used as an ingredient of curry spice. Incidents of IgE-mediated food allergy to fenugreek have been reported. Coincidence with allergy to peanut, a major food allergen, seems to be common suggesting a rather high rate of cross-reactivity. OBJECTIVE: Characterization of fenugreek allergens using patient sera and mass spectrometry-based proteomic analysis. METHODS: Allergenic fenugreek proteins were detected by immunoblotting, using sera from 13 patients with specific IgE to peanut and fenugreek. IgE-binding proteins were analyzed by peptide mass fingerprinting and peptide sequencing. RESULTS: A fenugreek protein quintet in the range from 50 kDa to 66 kDa showed high IgE-affinity, the protein at 50 kDa reaching the strongest signals in all patients. Proteomic analyses allowed the classification of several fenugreek proteins to a number of allergen families. Fenugreek 7S-vicilin and 11S-legumin were partly sequenced and revealed considerable homologies to peanut Ara h 1 and Ara h 3, respectively. The presence of a fenugreek 2S albumin and pathogenesis-related (PR-10) plant pollen protein was assumed by database searching results. CONCLUSION: In this study, individual fenugreek proteins were characterised for the first time. Observed homologies to major peanut allergens provide a molecular explanation for clinical cross-reactivity.

Effects of industrial processing on the immunogenicity of commonly ingested fish species.

BACKGROUND: Food-processing techniques may induce changes in fish protein immunogenicity. Allergens from >100 fish species have been identified, but little is known on the effects of processing on fish protein immunogenicity. METHODS: IgE binding of sera of patients allergic to fresh and processed (smoked, salted/sugar-cured, canned, lye-treated and fermented) cod, haddock, salmon, trout, tuna, mackerel and herring and of hydrolysates based on salmon and whiting was investigated using immunoblot and inhibition ELISA. RESULTS: Parvalbumin oligomers were identified using monoclonal and polyclonal antibodies. IgE binding was seen in most sera at 12-14 kDa (parvalbumin), and at 17-60 kDa for all fish except tuna. Changes in IgE binding appeared to reflect altered parvalbumin monomers and oligomers. Smoked haddock, salmon and mackerel had increased IgE binding and novel bands at 30 kDa. Chemically processed cod, salmon, trout and pickled herring had reduced or abolished IgE binding. The serum of 1 subject, however, had increased IgE binding to these products and also inhibition of binding by both fish hydrolysates to their constituent fish species. CONCLUSION: Process-induced changes in fish protein immunogenicity were more dependent on process rather than species, although individual responses varied. Changes in the allergenicity of a product may depend on the net effect of processing on parvalbumin oligomerization patterns, which may also vary in different species. Chemical processes generally caused loss in IgE-binding activity, though sensitization may occur to modified or degraded rather than intact peptides as shown by increased binding by chemically processed fish and hydrolysates in 1 subject. The clinical significance of these findings remains to be established.

Quantitative sandwich ELISA for the determination of tropomyosin from crustaceans in foods.

The ubiquitous muscle protein tropomyosin has been identified as the major shrimp allergen and is suggested to be a cross-reacting allergen. Previously, only a few methods for the detection of tropomyosin in food have been published. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of tropomyosin from crustaceans in foods has been developed and validated. A polyclonal rabbit antitropomyosin capture antibody and the biotinylated conjugate of the same antibody for detection were the basis for the ELISA, which was specific for crustaceans. The ELISA was able to quantitate tropomyosin in various food matrixes, had a detection limit of 1 microg/g, and cross-reacted to some extent with cockroach. Recoveries ranged from 63 to 120%, and the intra and interassay coefficients of variation were <6 and <14%, respectively.

Extractability, stability, and allergenicity of egg white proteins in differently heat-processed foods.

Hen's egg white protein is a major cause of food allergy, and a considerable number of countries have introduced labeling directions for processed food products. To control compliance with these regulations, analytical assays for the detection of egg in manufactured foods have been developed. In this study, we have tested the performance of 3 commercially available kits for quantitative egg analysis using 6 model heat-processed foods. The 3 assays worked well under standard conditions with soluble egg white proteins, but only the kit using a denaturing-reducing extraction buffer detected egg in complex heat-treated food matrixes. The differently extracted food samples were further used to evaluate the stability and allergenicity of the egg white allergens ovalbumin, ovomucoid, ovotransferrin, and lysozyme with polyclonal anti-egg antibodies and sera of 6 patients with egg allergy. It could be shown that differences in egg protein extractability have a significant impact on the interpretation of study results.

Sterol demethylation inhibitor fungicides as disruptors of insect development and inducers of glutathione S-transferase activities in Mamestra brassicae.

To study physiological and biochemical effects of demethylation inhibitor (DMI) fungicides on non-target insects, larvae of the cabbage moth, Mamestra brassicae L., were exposed orally to propiconazole, (R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole (100, 200 and 600 mg L(-1)) and fenpropimorph, (+/-)-cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl] 2,6-dimethylmorpholinc (10, 100, 200 and 600 mg L(-1)) in a semi-synthetic diet. Ten mg L(-1) of fenpropimorph reduced larval weight and induced in vitro glutathione S-transferase activity. Reduced larval and pupal growth rate, reduced survival, prolonged developmental time, and altered patterns of larval survival and adult emergence were found for one or both fungicides in at least one of the concentrations tested. The results suggest, that although the use of agricultural fungicides is generally regarded as of minor ecotoxicological consequence for insects, feeding on DMI-treated crops may influence insect fitness, and may also leave them susceptible to pesticide treatments or to residues of pesticides and other pollutants in their food. Standard methods to detect such effects should be developed for use in the environmental risk assessment of these products.

Casein-specific immunoglobulins in cow's milk allergic patient subgroups reveal a shift to IgA dominance in tolerant patients.

Differences in casein-specific immunoglobulin (Ig) G-subclass and IgA serum levels between reactive and tolerant patients may hint at the immunopathogenesis during tolerance development in cow's milk allergy (CMA). alpha-, beta- and kappa-casein-specific IgG(1), IgG(4), IgE and IgA serum levels were compared in clinically reactive and tolerized IgE-mediated (n = 15) and non-IgE-mediated (n = 14) CMA with delayed gastrointestinal symptoms, using enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques. The median anti-casein IgE levels in clinically reactive IgE-mediated CMA patients (n = 9) were 140- to 180-fold higher than in tolerized patients (n = 6) and 160- to 200-fold higher than in controls (n = 10). Median alpha-, beta- and kappa-casein-specific IgG(1) and IgG(4) levels were nine- to 60-fold higher in reactive patients and five- to 60-fold in tolerized patients. Clinical tolerance in IgE-mediated CMA was thus associated with decreased casein-specific IgE, IgG(4) and IgG(1), whereas serum IgA anti-alpha -, beta- and kappa-casein remained practically unaltered. Tolerized cow's milk protein (CMP)-sensitive atopic dermatitis had, in particular, decreased kappa-casein-specific IgG(1) levels, compared with clinically reactive patients. The ELISA levels to immunoblot correlation profile for the alpha-, beta- and kappa-casein-specific IgE suggested that the IgE-mediated CMA patients predominantly reacted to tertiary alpha- and beta-casein epitopes whereas the IgE in non-IgE-mediated patients reacted to linearized alpha-, beta- and kappa-casein epitopes. Clinical tolerance in non-IgE-mediated CMA patients (n = 9) was associated with a four- to 10-fold decrease in casein-specific IgE levels, accompanied by a five- to eightfold decrease in IgG(1) and five- to 60-fold decrease in IgG(4) levels, whereas casein-specific IgA levels remained unaltered. Thus, tolerance in both patient groups was characterized by a generalized decreased humoral immune response to caseins, which induced a functional shift to IgA dominance.

Memory T cell proliferation in cow's milk allergy after CD25+ regulatory T cell removal suggests a role for casein-specific cellular immunity in IgE-mediated but not in non-IgE-mediated cow's milk allergy.

BACKGROUND: Previously reported increased lymphocyte proliferative responses in cow's milk allergy (CMA) may have been influenced by the lipopolysaccharides (LPS) which contaminate most commercial cow's milk protein (CMPs). Moreover, peripheral blood mononuclear cells (PBMC) contain both B cells, CD45RA+ naïve T cells, CD25+ regulatory T cells (Tregs) in addition to antigen-specific CD45RA- memory T cells. METHODS: PBMC from clinically reactive and tolerised patients with IgE- and non-IgE-mediated CMA were depleted of CD45RA+ T cells and putative CD25+ Tregs. The proliferative index to LPS-depleted alpha-, beta- and kappa-casein and beta-lactoglobulin was compared in the memory T-cell-enriched, Treg-depleted PBMC and in bulk PBMC. RESULTS: Clinically reactive IgE-mediated CMA patients had increased responses to caseins only. Tolerised patients, particularly those with atopic dermatitis, had decreased responses to kappa-casein which were restored after Treg depletion. Interleukin-4 and interferon-gamma were generally not detected in the culture supernatants. No differences were seen between reactive and tolerant delayed non-IgE-mediated CMA patients. CONCLUSIONS: Proliferative responses to alpha-, beta- and kappa-caseins (but not beta-lactoglobulin) were observed in clinically reactive IgE-mediated CMA patients only. A markedly decreased proliferative response to kappa-casein in tolerised IgE-mediated CMA patients with atopic dermatitis, which was abrogated by Treg depletion, suggested a role for kappa-casein in tolerance induction. Non-IgE-mediated CMA patients had no increased proliferative response to any milk proteins.

Monoclonal antibodies against the candidate lupin allergens alpha-conglutin and beta-conglutin.

BACKGROUND: The ingestion of dietary products containing sweet lupin (such as Lupinus albus or Lupinus angustifolius) has been reported to cause IgE-mediated allergic reactions. Recent studies have indicated lupin globulins as important IgE binding proteins. The aim of the present study was to generate and characterize monoclonal antibodies (mAbs) against lupin seed proteins. METHODS: Mice were immunized with a protein isolate from L. albus and mAbs were obtained by hybridoma techniques. Albumins and globulins were extracted, and the globulin fraction was separated further into conglutins by anion exchange chromatography. Specificities, binding patterns and applications of the mAbs were investigated by immunochemical methods. RESULTS: Five mAbs were produced: Lu11 (an IgG2b antibody), Lu8, Lu18, Lu34 and Lu35 (all IgM antibodies). The mAbs reacted strongly with protein isolates from both L. albus and L. angustifolius. All mAbs are directed towards the lupin globulin fraction; Lu11 and Lu18 recognize alpha-conglutin, while Lu8, Lu34 and Lu35 recognize beta-conglutin. In addition, Lu11 inhibited the binding of IgE from patients with positive skin prick tests to lupin proteins in a competitive ELISA by approximately 30%. Furthermore, preliminary results show that Lu11 can be used to develop a sensitive method for the detection of alpha-conglutin in foods. CONCLUSIONS: Lupin globulins are immunogenic and alpha-conglutin is a potential allergen. This is the first study describing mAbs against the candidate lupin allergens, emphasizing the importance of additional studies on conglutins in lupin allergy.

Changes in humoral responses to beta-lactoglobulin in tolerant patients suggest a particular role for IgG4 in delayed, non-IgE-mediated cow's milk allergy.

The major cow's milk allergen beta-lactoglobulin (beta-LG) is relatively resistant to enzymatic degradation and may therefore be involved in non-immunoglobulin (Ig)E-mediated cow's milk allergy (CMA) with delayed gastrointestinal symptoms. Serum levels of beta-LG-specific IgG(1), IgG(4), IgE, and IgA were compared in clinically reactive and tolerized IgE-mediated and non-IgE-mediated CMA with delayed gastrointestinal symptoms (n = 29) and controls (n = 10). Tolerance was associated with decreased beta-LG-specific IgE, IgG(1), and IgG(4) levels in both patient groups. However, the significantly increased beta-LG-specific IgG(4) levels in clinically reactive non-IgE-mediated CMA patients, and its median 36-fold reduction in tolerant patients, suggested a possible immunopathological role for IgG(4) in delayed CMA. Similarly, the significantly increased beta-LG-specific IgE levels in IgE-mediated CMA patients were decreased 44-fold in tolerant patients. The tolerant patients had apparently shifted the humoral immune response from a beta-LG-specific IgE- and/or IgG(4)-dominated immune response to an IgA-dominated immune response as the IgA/IgE or IgA/IgG(4) ratios increased 90- and 15-fold in tolerant IgE-mediated-, and non-IgE-mediated CMA patients, respectively. Thus, the marked difference in beta-LG-specific Ig ratios suggested a tolerance-induced inhibition of a Th(2)-type of immune response with significantly increased IgA dominance in both CMA patient groups.

Quantitative sandwich ELISA for the determination of lupine (Lupinus spp.) in foods.

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.

A case of peanut cross-allergy to lupine flour in a hot dog bread.

BACKGROUND: In a case monitored by the Norwegian National Register for Severe Allergic Reactions to Food, a patient with peanut allergy experienced an allergic reaction after eating a particular brand of hot dog bread. The aim of this study was to identify the eliciting allergen. METHODS: Extracts from the hot dog bread and reference material from peanut, lupine and lupine-fortified food products were analysed by immunochemical methods with patient serum and a new polyclonal anti-lupine antibody. RESULTS: Evidence could be provided that the hot dog bread contained proteins from lupine but not from peanut. CONCLUSION: Crossed peanut-lupine allergy can have clinical significance. A peanut-allergic patient reacted against hidden lupine protein in a hot dog bread. Presented with our results, the producer confirmed the use of lupine flour and changed the ingredient list.