Sera of atopic dogs
To examine IgE reactivity in atopic dogs, we obtained sera from 179 dogs that were diagnosed with atopic dermatitis based on the criteria by Willemse  and Prelaud et al  among dogs visiting Fujimura Animal Hospital (Osaka, Japan). Twenty samples from laboratory dogs were used as negative controls. The dogs were housed indoors as experimental laboratory animals and had never been exposed to fish antigens. None of the laboratory dogs exhibited signs of atopic dermatitis. Enzyme-linked immunosorbent assay (ELISA) for parvalbumin or collagen from salmon (Atlantic salmon; Salmo salar), sardine (Japanese pilchard; Sardinops melanostictus), and mackerel (Chub mackerel; Scomber japonicus) were performed using individual sera of nine negative control dogs that provided a large amount of serum. All sera were stored at −80°C before use. Oral informed consent was obtained from the dog owners. All experimental procedures were carried out in accordance with Japanese law and approved by the animal care and user committee of Azabu University.
Food elimination and oral provocation tests
Among the 179 dogs with atopic dermatitis, 27 were confirmed to have cod reactivity by the oral provocation test after the elimination diet test; consent for food provocation test was obtained from the dog owners. These 27 dogs were fed commercial hydrolyzed protein diets as elimination diets for 6–8 weeks by the dog owners. When the veterinary physician recognized the complete resolution of the clinical signs during the food elimination test, the dog was admitted to the animal hospital and challenged with various cod ingredients, including grilled cod meat and cod-containing dog foods. The cod provocation test was discontinued immediately upon the relapse of the clinical signs including vomiting, diarrhea, erythema, pruritic urticaria, and conjunctival hyperemia .
Preparation of crude cod allergens
Cod is not only one of the most commonly consumed fish species in Europe and Japan  but also one of the most characterized fish species with allergen components . Thus, Pacific cod was purchased from a fish market in Japan to be used in the study. The fresh, raw meat of four fishes (500 µg) was homogenized in 500 µl phosphate-buffered saline (PBS, 10 mM pH 7.2) and rotated overnight at 4°C. After centrifugation at 21500 g for 5 min at 4°C, the supernatant was collected, and the protein was quantified using the BCA protein assay (Bio-Rad, Hercules, CA, USA).
Purification of parvalbumin, collagen, and tropomyosin
Fish parvalbumin  and collagen  were purified as described previously. Tropomyosin was purified from the ether powder by Bailey’s method with slight modification . Briefly, fish ether powder was stirred in a beaker with 75 ml extraction buffer containing 15 mM Tris HCl pH 7.6 (Sigma Aldrich, St Louis, MO, USA), 1 M KCl (Kanto Kagaku, Tokyo, Japan), and 2 mM dithiothreitol (Sigma Aldrich) overnight at 4°C. The extract was collected by centrifugation at 5400 g for 10 min at 4°C. The supernatant pH was adjusted to 4.5 with 1 N HCl to precipitate tropomyosin, and the precipitation was collected by centrifugation at 5400 g for 10 min at 4°C. The isoelectric precipitation was repeated once, and the precipitated material was dissolved in the extraction buffer. The supernatant after the extraction was collected by centrifugation and fractionated by ammonium sulfate at a concentration of 50%. The sample precipitated by ammonium sulfate was dissolved and dialyzed against PBS. The obtained protein extracts were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).
SDS-PAGE and immunoblotting
Among the 20 atopic dogs with IgE sensitivity to crude cod allergens that did not react to parvalbumin or collagen, sera were used from two dogs (no. 34 and no. 128) that provided a large amount of sera and reacted to crude cod allergens but not cod parvalbumin or collagen, as confirmed with the provocation test using cod-containing foods (Table 1). SDS-PAGE was performed according to the method of Laemmli . Precision plus protein standards (Bio-Rad) were used as molecular-mass markers. Protein components were electrophoretically separated using 5%–20% gradient polyacrylamide gels, and proteins were visualized either by Coomassie brilliant blue R250 (Bio-Rad) or transferring onto polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA). Immunoblotting was performed as described previously . IgE in patient dog sera were used as primary antibodies, which were diluted 1:10 in tris buffered saline containing 0.1% Tween-20 and 5% nonfat dried milk. Mouse monoclonal anti-dog IgE antibodies (0.5 μg/ml) were used as secondary antibodies . Detection was performed using an enhanced chemiluminescence immunoblotting detection reagent (GE Healthcare).
Liquid chromatography (LC)-tandem mass spectrometry (MS/MS)
For LC- MS/MS, the protein bands detected by immunoblotting were excised, and in-gel digestion was performed with 0.5 mg N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Promega, Madison, Wisconsin) at 37°C for 16 h. Tryptic digests were acidified using formic acid (pH < 2.0) and centrifuged at 21500 g for 15 min. The supernatants were analyzed using high-performance LC (Advance System; AMR, Tokyo, Japan) connected to an electrospray ionization triple quadrupole mass spectrometer (4000 QTRAP; AB Sciex, Framingham, MA, USA). Extracts were injected into a reversed-phase column (electrospray ionization column [octa decyl silyl]; particle inner diameter, 75 mm; length, 100 mm; diameter 3 mm; LC Assist, Tokyo, Japan) that was eluted with a 5%–45% gradient of acetonitrile containing 0.1% formic acid for 60 min at 300 nl/min. Ionization was performed using an ion-spray voltage of 2000 V at a capillary temperature of 200°C. The mass spectrometry instrument was operated in the positive ion mode over the range of 450–1200 m/z. The MS/MS spectra were obtained in the enhanced production scan mode, and two higher-intensity peaks in each mass spectrometry scan were chosen for collision-induced dissociation.
The MS/MS data were used to search in entries under the Liza aurata category of the UniProt database using the Mascot peptide search engine. An MS tolerance of 1.0 Da for-precursor ion and an MS/MS tolerance of 0.8 Da were set as windows of processing parameters for matching peptide mass values.
Fluorometric ELISA for allergen-specific serum IgE
Specific IgE levels to cod crude allergens were measured using a fluorometric ELISA as previously described . A microplate (NUNC Immuno Plate Maxisorp F96; Nalge Nunc International, Roskilde, Denmark) was coated with crude cod extracts (10 µg/ml) or the purified allergens (parvalbumin, collagen, or tropomyosin; 1 µg/ml) at 4ºC overnight. After washing, the plate was incubated with diluted sera (1:10) at room temperature for 3 h. The plate was then washed and incubated at 4ºC overnight with a mouse monoclonal anti-dog IgE antibody (0.5 µg/ml) . After washing with PBS containing 0.1% (v/v) Tween 20 (PBS-T), the plate was incubated with a biotinylated rat monoclonal anti-mouse IgG1 (Zymed Laboratories, San Francisco, CA, USA) at room temperature for 1 h. After washing, the plate was incubated with b-D-galactosidase-conjugated streptavidin (Zymed Laboratories) at room temperature for 1 h. After the final washing, the plate was incubated with 0.1 mM 4-methylumbelliferyl- b-D-galactopyranoside (Sigma Aldrich) at 37ºC for 2 h. The enzymatic reaction was stopped with the addition of 0.1 M glycine-NaOH (pH 10.2). The fluorescence intensity was measured as fluorescence units (FU) on a microplate fluorescence reader (Fluoroskan; Flow Laboratories, McLean, VA, USA). The absorbance was measured at 355 nm with a 460 nm reference filter. All the washing steps were performed three times for 5 min in PBS-T. The cutoff value was determined as the average + three standard deviations (SDs) of FU in serum samples of 20 dogs used as negative controls. All tests were performed in triplicate.