No abstract

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin beta (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal sl). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal sl was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST- and ELISA-inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA-library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the beta-lineage of parvalbumins and include the entire coding region.

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.

No abstract

During the last decade, cases of the fish parasite Anisakis simplex infection and allergy in human have increased in countries with high fish consumption. Our aim was to perform an extended seroprevalence study of anti-IgE antibodies against this parasite in Norway, one of the high fish-consuming countries. At the Department of Immunology and Transfusion Medicine and the Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, two main groups of anonymized serum samples were collected; the first (n = 993) from recently recruited blood donors (designated 'BDO') and the second (n = 414) from patient with total IgE levels ≥1000 kU/l (designated 'IGE+'). The sera were analysed by the ImmunoCAP â method for total IgE and IgE antibodies against A. simplex, house dust mite (HDM), shrimp, cod, crab, brine shrimp and shrimp tropomyosin. The A. simplex positive sera were further tested by an enzyme-linked immunosorbent assay (ELISA) method, which uses 2 recombinant (r) major allergens, rAni s 1 and rAni s 7 as target antigens. SDS-PAGE and Western immunoblotting analyses were also performed. Whereas the prevalences by ImmunoCAP â were 0.4% and 16.2% in the BDO and IGE+ groups, respectively, analyses with recombinant allergens showed only 0.0% and 0.2%. Cross-reactivity and immunoblotting analyses suggested that most of the ImmunoCAP â positive sera were probably false-positive due to cross-sensitization to shrimp and HDM. However, positivity due to other A. simplex antigens should also be considered. Compared with other high fish-consuming countries, we observed a very low seroprevalence of anti-Anisakis IgE antibodies in a Norwegian population.

No abstract

The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1. Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin‐encoding regions, were cloned, revealing transcripts from two different parvalbumin genes. In the present study, the protein‐coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET‐19b). Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host. The recombinant products in the soluble fraction were purified using the His tag‐purification system and analysed on Western blots with anti‐salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish. Both recombinant products (His10‐14.1 and His10‐24.1) reacted positively with salmon parvalbumin‐specific immunogloblin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish‐allergic patients. The allergenicity of His10‐14.1 was confirmed using enzyme‐linked immunosorbent assay (ELISA). The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library.

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323‐339 has been reported to be responsible for 25–35% of isolated BALB/c mouse T‐cell response to intact OVA. An investigation of whether OVA and OVA 323‐339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight‐week‐old BALB/c mice were randomly divided into three groups: OVA, OVA 323‐339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 μg OVA or OVA 323‐339 absorbed on 300 μg Alum, or saline; on days 21–23, all groups were challenged intranasally with either 20 μl of 1% OVA, 1% OVA 323‐339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323‐339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA‐specific IgE were observed in OVA mice when compared to saline control. OVA 323‐339 mice showed higher serum OVA‐specific IgE, OVA 323‐339‐specific IgE, IL‐4 and lower IFN‐γ similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323‐339 mice, while the similar proliferative response to OVA 323‐339 was only observed in the splenocytes of OVA 323‐339‐sensitized and challenged mice. Although OVA 323‐339 induced a Th2‐like response in the mouse model as did OVA, OVA 323‐339 has clearly limited immunogenic potency to activate OVA‐sensitized and challenged mice splenocytes, unlike OVA.