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日本調理科学会誌（J.Parvalbumin Cookery Antigenicity Sci. Jpn.） Vol. and Allergenicity 52，No. 3，147～158（2019） of 127 Species of Fi〔Originalsh paper〕 Characterization of Parvalbumin in 127 Species of Fish by Enzyme-linked Immunosorbent Assay Using Monoclonal Anti-frog Parvalbumin IgG Antibody and Serum IgE from an Allergic Patient Kaori Kurata*§ Akira Dobashi* Kazuyuki Kurihara** Yasuharu Itagaki*** Parvalbumin（PA） is a major allergen of fish allergy, and its content varies between different species of fish. In this study, we evaluated the antigenicity of PA in 127 commercially available fish species by enzyme-linked immu- nosorbent assay（ELISA）. First, we evaluated the antigenicity and allergenicity of 12 species of fish belonging to the Salmonidae family by ELISA with monoclonal anti-frog PA IgG antibody and serum IgE from an allergic patient. Among the 12 salmonid fish, shiro-zake（chum salmon） showed the least antigenicity, and landlocked fish generally showed more antigenicity than sea-run fish. Trout and landlocked species that are not among the specified food ingredients for labeling in Japan were higher in antigenicity and allergenicity than the salmon that has traditionally been eaten so far, indicating that salmon-reactive patients should be careful with these fish. The screening of 124 species of fish with the monoclonal anti-frog PA IgG antibody revealed that anago（common Japanese conger） had high antigenicity. Among the Osteichthyes, the antigenicity was low in shiro-saba-fugu（green rough-backed puffer） and shira-uo（ice fish）. Among the Chondrichthyes, the antigenicity of ma-kasube（mottled skate）, yoshikiri-zame（blue shark）, and kawa-yatsume（Japanese lamprey） was also low. The PA content of four fish species, as quantified by ELISA using purified PA as a standard, was 17.6 mg/g in shiro-guchi（white croaker）, 6.1 mg/g in shiro-zake（chum salmon）, 4.9 mg/g in toki-shirazu（chum salmon） and 5.9 mg/g in shishamo （shishamo smelt）. Keyword：food allergy, fish allergy, parvalbumin, Chondrichthyes, ELISA （Müntener et al., 1995）. The muscles of fish and amphibi- INTRODUCTION ans contain relatively large quantities of PA （Gerday, Fish allergy can cause stress and unpleasant symptoms, 1982）, and it has been clearly demonstrated that PA such as itching, vomiting, diarrhea, and life-threatening shows antigenic cross-reactivity between different species anaphylactic shock, among not only adults, but also chil- of fish and amphibians （Hilger et al., 2004）. dren（Sichere, 2011; Hajab and Selamat, 2012）. Although Fish species can be divided into two main groups: bony there are more than 34,000 different species of fish fish and cartilaginous fish. Most edible fish are bony fish described in the FishBase database（www.fishbase.org）, that belong to the class Osteichthyes, whereas sharks and the consumption of fish depends heavily on regional avail- rays are cartilaginous and belong to the class Chondrich- ability. However, less than 0.5% of all known fish species thyes. Osteichthyes can be further divided into 45 orders; have been analyzed for their allergens on the molecular the most widely consumed Osteichthyes around the world level, and the ones that have been analyzed have shown belong to the orders of Clupeiformes（herrings and sar- unexpectedly large diversity（Sharp and Lopata, 2014）. dines）, Salmoniformes （salmons and trouts）, Cyprini- Parvalbumin（PA） is known to be a major allergen of formes （carps）, Gadiformes （cods）, and Perciformes fish allergy. It is a 10- to 12.5-kDa protein that is water （perches, mackerels, and tunas） （Sharp and Lopata, soluble and binds calcium（Arif, 2009）. PA is found in the 2014）. In Japan, there are close to 300 kinds of edible fish sarcoplasm of all vertebrates, including humans, and plays （Itagaki, 2011）, and it is sometimes difficult to distinguish an important role in the relaxation of muscle fibers biologically related species from their common names. For example, the fish known as blackthroat seaperch in * Tokyo University of Pharmacy and Life Sciences ** Kanagawa Children’s Medical Center English is called “aka-mutsu” or “nodo-guro” in Japan; *** Kanagawa Prefectural Institute of Public Health the term used differs between different areas of Japan. In Present affiliation: Hokkaido Bunkyo University § Inquiry Education and Research Institute of Information Science, addition, “aka-mutsu” and “mutsu” belong to the order Tokyo University of Pharmacy and Life Sciences Perciformes, but the former belongs to the Acropomatidae 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan family, and the latter belongs to the Scombropidae family, TEL 042（676）3095 FAX 042（670）7067 E-mail：[email protected] which may be confusing since both have “mutsu” in their （147） 19 日本調理科学会誌（J. Cookery Sci. Jpn.） Vol. 52 No. 3（2019） names. Stems like “-dai” and “-aji”, which are used in zake（chum salmon）, toki-shirazu（chum salmon）, and Japanese, are also confusing since they are not related to karafuto-masu（pink salmon）. Three of these species of any biological classification system, and the stems were salmonid fish （niji-masu, masunosuke, and toki- attached based on the cultural history of the fish, and may shirazu） were not among the 124 species included in the indicate their culinary uses or whether they are consid- screening test. The 23 species observed with SDS-PAGE ered to be preferable and/or delicious. were anago（common Japanese conger）, yoshikiri-zame The main aim of our research is to determine the kinds （blue shark）, ma-kasube（mottled skate）, kawa-yatsume of fish that can be eaten by allergic patients, who have （Japanese lamprey）, shira-uo （ice fish）, hotei-uo had allergic reactions to certain fish species; this is impor- （smooth lumpsucker）, noro-genge（porous-head eelpout）, tant because the consumption of fish can help individuals, kihada （yellowfin tuna）, kamasu-sawara （wahoo）, especially pediatric patients, to maintain a healthy dietary hatahata（Japanese sandfish）, shiro-saba-fugu（rough- balance（Rance et al., 2005）. In Japan, marine products backed puffer）, manbou （ocean sunfish）, ma-kajika account for 40% of the dietary animal protein intake, （great sculpion）, nore-sore （juvenile conger）, nishin according to the Food Balance Sheet for fiscal year 2008, （pacific herring）, wakasagi（Japanese smelt）, shisyamo which was prepared by the Ministry of Agriculture, For- （shisyamo smelt）, ayu（Japanese trout）, komai（saffron estry and Fisheries. The Japanese Pediatric Guideline for cod）, suketou-dara（Alaska pollack）, hokke（akta mack- Food Allergy 2016 of the Japanese society of pediatric erel）, hakkaku （sailfin poacher）, kurogashira-karei allergy and clinical immunology is pursuing “What can we （cresthead flounder）, and karei（righteye flounder）. do to help allergic patients eat?”. Recently, oral immuno- The 127 kinds of commercially available fish were therapy has been offered to patients with allergies to egg, mainly purchased from fish markets, and the edible por- milk, and wheat, items for which tolerance can be rela- tions（fillets） were separated from the whole fish and tively easy to attain（Ebisawa, 2018）. If some fish species stored at -20℃. Each thawed fillet was then divided into among the over 300 kinds of edible fish can be identified eight pieces, from the head to the tail; 10 g from each as having a low PA content and/or low allergenicity, then piece were combined and chopped, then homogenized for it may be possible for mild allergy sufferers to eat those 1 min in a 5-fold volume of 10 mM phosphate buffer （PB, fish, or the fish could be applied in oral immune tolerance pH 7.0）. The mixture was incubated at 4℃ overnight, therapy as a hypoallergenic fish. and then centrifuged at 20,000×g and 4℃ for 30 min. In this study, we screened the reactivity of monoclonal The supernatant was filtered through a 5-μm syringe fil- anti-frog PA IgG antibody against extracts of 127 kinds of ter to obtain the experimental fish extracts. fish that are commercially available on the Japanese mar- Experimental serum ket, and we compared the antigenicity and allergenicity of The experimental serum, AK-9, was provided by a 12 kinds of salmonid fish using patient serum. Further- patient with fish allergy, who showed a score of 5 for more, we attempted to clarify the differences in PA con- salmon on the radioallergosorbent test. The patient was a tent between different fish species by measuring the PA 3 year old girl who had experiences of allergic symptoms content of fish, such as white croaker, which is often used by eating aji no himono（dried horse mackerel） and egg. as a raw material for processed fish products, and chum This patient serum was obtained with the informed con- salmon, which is widely eaten. sent of the patient, and was used after confirming that it reacts to the PA of shiro-zake, but not to the collagen of MATERIALS and METHODS shiro-zake. The control serum, VT-1, was provided by a Fish materials person with no allergy. Our study used a total of 127 species of fish; a list of the Evaluation of IgG reactivity （antigenicity） by an names of the fish species is shown in Appendix 1. The 12 enzyme-linked immunosorbent assay （ELISA） using species of salmonid for which the antigenicity and allerge- monoclonal anti-frog PA IgG antibody nicity were evaluated were: itou（Japanese huchen）, niji- Diluted fish extracts（1:800 and 1:1600, diluted with 5 masu （rainbow trout）, salmon trout, hime-masu mM Na2CO3-20 mM NaHCO3（pH 9.5）） were coated onto （kokanee）, yamame（landlocked trout）, iwana（whites - 96-well flat-bottom polystyrene microtiter plates potted char）, masunosuke （king salmon）, beni-zake （Maxisorp; Nalge Nunc International, NY, USA） at 50 μl/ （sockeye salmon）, sakura-masu（cherry salmon）, siro- well. After incubation for 1 h at 37℃, the plates were 20 （148） Parvalbumin Antigenicity and Allergenicity of 127 Species of Fish washed three times with 0.05% Tween20 containing Laboratories Inc.） and standard BSA.

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