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 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 , the antigenicity was low in shiro-saba-fugu（green rough-backed puffer） and shira-uo（ice fish）. Among the , the antigenicity of ma-kasube（mottled ）, 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 ）.

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..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 fish allergy. It is a 10- to 12.5-kDa protein that is water （perches, , and ） （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

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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 ）, 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 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 ） 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. Four dilutions of a phosphate-buffered saline（PBS, pH 7.0） using an auto- BSA standard were prepared; they contained from 0.2 to matic plate washer; the plates were sealed with plate- 0.8 mg/ml of BSA. The experimental fish extracts were sealing tape during each incubation. The wells were diluted 10- or 20-fold with buffer. Next, 25 μl of the blocked with 300 μl/well of Dulbecco’s PBS（DPBS） con- diluted extracts, 125 μl of Reagent A, and 1 ml of Reagent taining 1% bovine serum albumin（BSA） for 1 h at 37℃. B were mixed and stirred in a microtube, then allowed to The wells were then incubated with 50 μl/well of mouse stand for 5 min. Absorbance was measured at 750 nm monoclonal anti-frog PA antibody（Sigma-Aldrich Co., using a SmartSpec Plus Spectrophotometer （Bio-Rad MO, USA; 1:4000, diluted with DPBS containing 0.1% Laboratories Inc.）. The amount of protein per 1 g of fish BSA） for 1 h at 37℃, followed by incubation with 50 μl/ meat was calculated based on the obtained absorbance. well of horseradish peroxidase-conjugated rabbit anti- Sodium dodecyl sulfate polyacrylamide gel electrophore- mouse IgG antibody（ICN Pharmaceuticals Inc., CA, USA; sis（SDS-PAGE） analysis 1:4000, diluted with DPBS containing 0.1% BSA） for 1 h The fish extracts were diluted to a protein concentra- at 37℃. After a final incubation with 50 μl/well of tetra- tion of 1 mg/ml with SDS sample buffer with 2-mercapto- methylbenzidine substrate（Pierce Biotechnology Inc., IL, ethanol, and then applied with 10 μl/lane to a 15% USA） at room temperature, the reaction was stopped by e-PAGEL polyacrylamide gel（ATTO, Tokyo, Japan） for

the addition of 100 μl/well of 2 M H2SO4, and the absor- separating the proteins. Pre-stained SDS-PAGE broad bance was measured at 450 nm using a microplate reader range standards（Bio-Rad Laboratories Inc.） were used as （Bio-Rad Laboratories Inc., CA, USA）. references. SDS-PAGE was performed using pageRun Evaluation of the IgE reactivity（allergenicity） of 12 （ATTO） by applying 20 mA/gel for 85 min. The gels species of fish belonging to the Salmonidae family by were stained with Coomassie brilliant blue. ELISA using serum from an allergic patient Purification of PA from the edible portion of fish meat

Diluted extracts（1:1000, diluted with 5 mM Na2CO3-20 Purification of PA from the edible portion of shiro-

mM NaHCO3（pH 9.5）） were coated onto 96-well flat- guchi（white croaker）, shiro-zake and toki-shirazu, and bottom polystyrene microtiter plates（Maxysoap; Nalge shisyamo was performed according to the following pro- Nunc International） at 50 μl/well. After incubation for 2 h cedures. The fish meat was diluted 4-fold with 10 mM PB at 37℃, the plates were washed five times with 0.05% （pH 7.0） and then homogenized. The mixture was Tween20 in PBS （PBST, pH 7.0） using an automatic allowed to stand at 4℃ for 3 h, and then centrifuged at plate washer; the plates were sealed with plate-sealing 20,000×g and 4℃ for 40 min. The supernatant was fil- tape during each incubation. The wells were blocked with tered using a 0.5-μm syringe filter to obtain the fish 300 μl/well of Starting Block （PBS） Blocking Buffer extract. （Pierce Biotechnology Inc.） for 1 h at 37℃ before being The extract was subjected to chromatographic analysis incubated again with 50 μl/well of patient serum（diluted under the following conditions: column, Sephacryl-S-100 1:50 with PBST containing 5% blocking buffer） for 1 h at HR（GE Healthcare Life Sciences, NJ, USA; 2.6×100 cm）; 37℃, and then 4℃ overnight. PA-reactive serum（AK-9） eluent, 10 mM PBS（pH 7.0）; flow rate, 20 to 30 ml/h; or control serum（VT-1） was used as the primary anti- column temperature, 4℃; and fractional volume, 6.0 ml. body, and the secondary antibody was horseradish perox- The combined eluate was further chromatographed idase-conjugated goat anti-human IgE antibody （ICN with a Source 15RPC（GE Healthcare Life Sciences; 4.6× Pharmaceuticals Inc.; 1:10000, diluted in the same buffer）. 100 mm） column using 0.1% trifluoro acetic acid in 70% Secondary antibody at 50 μl/well was added and allowed AcCN for column equilibration, followed by linear gradient to react for 1 h at 37℃. Next, 50 μl/well of SuperSignal elution from 0% to 100%. The equilibration was performed Chemiluminescent Substrate（Pierce Biotechnology Inc.） with a flow rate of 1.0 lm /min and a fractional volume of was added, and the mixture was incubated at room tem- 1.0 ml. The elution was performed with a flow rate of 2.0 perature for 5 min. Subsequently, the relative light units ml/min and a fractional volume of 0.5 ml. were measured at 425 nm. The combined eluate was further chromatographed as Protein quantification necessary under the following conditions: column, The total protein content was measured by the Lowry Superdex 75（GE Healthcare Life Sciences; 1.0×30 cm）; method with the Bio-Rad DC Protein Assay Kit（Bio-Rad flow rate, 1.0 ml/min; fractional volume, 1.0 ml; and elu-

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tion condition, 10 mM PBS（pH 7.0）. fish. We grouped three pairs of landlocked and sea-run After chromatographic separation, fractions containing fish: hime-masu and beni-zake, yamame and sakura- PA were identified by ELISA using monoclonal anti-frog masu, and niji-masu and salmon trout, respectively. PA antibody, as described in the previous section. The amount of protein contained in the extracts Measurement of PA content by ELISA using purified obtained from 9 fish species was shown in the Appendix PA 1. The amount of protein of niji-masu, masunosuke, and A standard curve was generated for the purified PA, as toki-shirazu, which were not listed in the Appendix 1, described above. The optimum dilution ratio of samples were 6.0 mg/ml, 9.8 mg/ml, and 10.1 mg/ml, respec- was selected from 1:800, 1:1600, and 1:3200, and 5 mM tively.

Na2CO3-20 mM NaHCO3（pH 9.5） was used as the dilu- Figure 1a shows a comparison of the antigenicity of the ent. The other procedures and reaction times were the 12 kinds of salmonid fish. The ELISA assay using mono- same as those described above in the “Evaluation of IgG clonal anti-frog PA IgG antibody revealed large differ- reactivity（antigenicity） by an enzyme-linked immunosor- ences in the antigenicity of the tested fish, and that the bent assay（ELISA） using monoclonal anti-frog PA IgG differences were species-dependent. The fish with the antibody” section. highest PA content was itou, followed by niji-masu and Statistical analysis salmon trout. Niji-masu is of the same species as salmon The ELISA measurements were performed in duplicate trout, but is a landlocked form of the fish. The remaining （allergenicity） or triplicate（antigenicity）, and the values landlocked species, i.e., hime-masu, yamame, and iwana, were expressed as the mean ± standard deviation（SD）.

Differences in antigenicity between chum salmon and (a) other salmonid species were assessed by one-way analy- 1.6 *

sis of variance（ p＜0.05）, and multiple-range comparisons 1.4 * *

were performed using the Bonferroni test. 1.2 * * Ethical approval 1.0 * The Ethics Committee of Kanagawa Prefectural 0.8 (450 nm) 0.6 *

Institute of Public Health（Kanagawa, Japan） approved OD * * this study（approval numbers: 2005, No. 5 for the approval 0.4 0.2 period of October 1, 2005 to March 31, 2008; and 2006, No. 0.0 5 for the approval period of November 1, 2006 to March 12345 6789 10 11 12

31, 2009）. (b) RESULTS and DISCUSSION 12 PA-reactive subject 10 Control subject

Screening survey of the IgG reactivity（antigenicity） x 100,000 425 nm) and IgE reactivity（allergenicity） of 12 species of fish 8 units ( belonging to the Salmonidae family 6 light Fish allergy sufferers are instructed to refrain from 4 ingesting any fish or marine products. In this study, 127 Relative 2 species gathered from all over Japan were exhaustively 0 studied for the purpose of identifying fish that may be 1234567 8 9 10 11 12 safely ingested by patients with allergies to fish meat. Fig. 1. The reactivities of IgG and IgE against 12 kinds of fish First, a comparative study was conducted to examine the belonging to the Salmonidae family by ELISA. IgG reactivity（antigenicity） and IgE reactivity（allerge - （a） IgG reactivity using monoclonal anti-frog PA IgG antibody. The nicity） of 12 fish belonging to the Salmonidae family with data of the landlocked fish are shown as striped bars. Asterisks indi- cate a significant difference when compared to chum salmon（ p＜ a focus on PA, which is the main allergen of fish. 0.05）.（b） IgE reactivity of serum from a fish allergy patient and Salmon that are edible in Japan can be broadly divided serum from an individual with no fish allergy. Bars in a and b: 1, into those born in rivers and living in the sea, and those itou; 2, niji-masu（landlocked form of 3）; 3, salmon trout; 4, hime- masu（landlocked form of 8）; 5, yamame（landlocked form of 9）; 6, that start and end life as freshwater fish. The former are iwana; 7, masunosuke; 8, beni-zake; 9, sakura-masu; 10, toki- called sea-run fish, and the latter are called landlocked shirazu; 11, karafuto-masu; and 12, shiro-zake.

22 （150） Parvalbumin Antigenicity and Allergenicity of 127 Species of Fish

showed higher antigenicity, followed by the sea-run fish anadromous salmon were subject to the labeling system, beni-zake and sakura-masu. Shiro-zake, toki-shirazu, there were many cases in which allergic patients con- and karafuto-masu had low antigenicity. Statistical analy- sumed landlocked trout and suffered an allergic reaction. sis showed that there was no significant difference in Although Kondo et al.（2009） and Shimakura et al.（2012） reactivity between shiro-zake, toki-shirazu, and karafto- have since reported data on the comparison of allergens in masu, and that other salmonid fish were significantly salmon and landlocked trout, the labeling regulations have more reactive than shiro-zake. not been changed to include landlocked salmon. Figure 1b shows a comparison of the allergenicity of the Similarly, the importation of salmon, such as 12 kinds of salmonid fish. All species of the salmonid fish masunosuke（king salmon）, has been on the rise in Japan showed reactivity with the IgE in the serum of the in recent years, but the landlocked species are not among salmon allergy patient. Allergenicity was highest in hime- the food ingredients that need to be labeled despite being masu and lowest in karafuto-masu. Fish with high anti- higher in antigenicity and allergenicity than the traditional genicity had high allergenicity, while those with low aller- salmon that has been eaten in Japan so far. Therefore, it is genicity had low antigenicity. necessary for salmon-reactive patients to be careful with Shiro-zake and beni-zake are salmon that have been imported salmon. In addition, itou and iwana are known eaten on a daily basis in Japan since ancient times. Shiro- as clear stream fish belonging to the Salmonidae family, zake is a sea-run fish, and like beni-zake, it is born in the but they have higher antigenicity and allergenicity than river, goes out to sea, and then returns to the river. The shiro-zake, and again, it is necessary for fish allergy antigenicity and allergenicity of these two salmon species patients, especially those with salmon allergy, to be care- were among the lowest in the 12 species of salmonid fish ful. examined in this study. Screening survey of the antigenicity of 124 species of Issues with allergy labeling for specified food ingredi- commercially available fish ents, as highlighted by the comparative study of 12 spe- The Osteichthyes class can be divided into 45 orders, cies of fish belonging to the Salmonidae family and Japanese people are thought to consume fish from Niji-masu, hime-masu, and yamame, which are all over 20 of these orders. Although only mackerel and known as landlocked forms of trout, showed higher antige- salmon need to be labeled for food allergies, cross- nicity and allergenicity among the experimental salmonid reactivity is known to occur in fish allergy, and people fish （Figure 1）. Similar results were obtained in previous who have experienced fish allergy should be careful not detailed comparisons of yamame and sakura-masu only with the fish species that caused the initial allergy, （Shimakura et al., 2012）, and hime-masu and zeni-zake but also with other fish species. Therefore, in this study, （Kondo et al., 2009）. 124 fish species were further examined comprehensively Kondo et al.（2009） proposed that the quality of PA by ELISA using monoclonal anti-frog PA antibody. was the same between yamame and sakura-masu, and We examined a total of 124 kinds of fish species, three that the difference in allergenicity between the two fish kinds from the Chondrichthyes class and 121 kinds from was due to differences in PA content. Fast muscles are the Osteichthyes class（Appendix 1）. The Chondrichthyes thought to be high in PA content, and the life history of examined included only Agnatha from three orders, i.e., the landlocked form of salmon may be closely related to Carcharhiniformes, , and Petromyzontiformes. the amount of fast muscle they contain. The Osteichthyes included fish from 17 orders, i.e., Since 2001, the Japanese Food Labeling Act has Anguilliformes （4 species）, Cluperiformes （6 species）, included salmon on the list of specified food ingredient for Cyprinformes （2 species）, Salmoniformes （9 species）, which labeling is recommended. In contrast, there is no （4 species）, Gadiformes （3 species）, recommendation for the labeling of landlocked trout Bericiformes （1 species）, Beloniformes （4 species）, despite salmon and trout belonging to the same species; Scorpeaniformes（13 species）, Mugiliformes（1 species）, the two fish only live in different habitats. Kondo et al. Gasterosteiformes（1 species）, Siluriformes（1 species）, （2009） pointed out that when the allergy labeling system Aulopiformes （2 species）, Zeniformes （1 species）, was established, there were no data for comparing the Perciformes（59 species）, Pleuronectiformes（5 species）, allergens between landlocked salmon and the sea-run/ and Tetraodontiformes（5 species）. The names of all of anadromous salmon species, and since only sea-run/ the experimental fish, which were numbered from sample

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1 to 124, the measured protein contents, and the optical Those showing a low OD value of 0.01 to 0.05 included density（OD） values are all shown in Appendix 1. The 12 species, i.e., nore-sore（sample 6）, wakasagi（sample average protein content was 39.2±14.8 mg/g of fish 25）, ayu（sample 27）, suketou-dara（sample 29）, hokke meat, which was calculated from 6.54±2.47 mg/ml. （sample 47）, ai-buri（blackbanded trevally, sample 61）, 1. Results of the three species classified as Chondrich- shiira（common dolphinfish, sample 74）, ira（tuskfish, thyes sample 84）, ma-saba （chub mackerel, sample 85）, For the Chondrichthyes, i.e., yoshikiri-zame（sample 1 kamasu-sawara（sample 89）, ishigaki-dai（parrot-bass, in Appendix 1）, ma-kasube （sample 2）, and kawa- sample 100）, and kurobishi-kamasu （snake mackerel, yatume（sample 3）, the protein content was 3.8, 2.7, and sample 114）. 7.3 mg/ml, respectively, and they showed no antigenicity. Antigenicity was not detectable in 11 species of 2. Results of the 121 species classified as Osteichthyes Osteichthyes, i.e., shira-uo（sample 28）, komai（sample The high-reactivity group of fish showing an OD value 31）, ma-kajika（sample 37）, hotei-uo（sample 49）, noro- of 1.5 or more included 6 species, i.e., anago（sample 5）, genge（sample 57）, kihada（sample 89）, hatahata（sam - ma-haze（yellowfin goby, sample 80）,hotaru -jyako（lan - ple 98）, karei（sample 116）, kurogashira-karei（sample ternbelly, sample 104）, ebo-dai（wart perch, sample 107）, 118）, shiro-saba-fugu（sample 120）, and manbou（sam - chikame-kintoki（longfinned bullseye, sample 112）, and ple 123）. shita-birame（, sample 119）. Those showing an Similar to the results of the studies done in the OD value of 1.0 to 1.5 included 18 species, including Salmonidae family, the OD values varied widely even unagi（Japanese eel, sample 4）, mamakari（big-eye sar- between fish of the same family. dine, sample 10）, konoshiro（threadfin shad, sample 11）, 3. Results of SDS-PAGE analysis katakuchi-iwashi（Japanese , sample 12）, koi Figure 2 shows the results of the SDS-PAGE analysis （carp, sample 14）, dojyo（loach, sample 15）, itou,（sample of 23 fish species that showed low antigenicity or no anti- 19）, salmon trout（sample 24）, sayori（Japanese halfback, genicity on the screening test using ELISA. sample 35）, matou-dai（john dory, sample 55）, kuro-dai The hotei-uo, noro-genge, hatahata, and kurogashira- （blackhead sea-bream, sample 72）, kuro-mutsu karei samples showed PA bands on SDS-PAGE, which （Japanese bluefish, sample 76）,hon -kamasu（barracuda, was interesting, because they did not show reactivity in sample 93）, mejina （greenfish, sample 94）, ishi-dai the ELISA screening. （barred knifejaw, sample 99）, isu-zumi（waigeu drum- The fish species with no bands in the molecular region mer, sample 106）, Japanese large-eye bream（ shiro-dai, predicted to be PA were the Chondrichthyes yoshikiri- sample 110）, and umadura-hagi（filefish, sample 122）. zame and kawa-yatume, and the Osteichthyes kihada,

(kDa) (kDa) 194.8 194.8 109.6 109.6 56.8 56.8

41.5 41.5

27.8 5 27.8 20.7 20.7

15.4 15.4

6.6 10 6.6

M P1 1234 5 6 7 8910 11 12 M P1 13 14 15 16 17 1819 20 21 22 23 P2

Fig.Figure 2. SDS-PAGE 2. SDS-PAGE of the the extracts extracts of 23 ofspecies 23 species of fish that of showedfish that low antigenicityshowed low or no antigenicity antigenicity on or the no ELISA antigenicity screening test.on theLane ELISA M, 15 molecularscreening weight marker test. (from bottom to top: 6.6, 15.4, 20.7, 27.8, 41.5, 56.8, 109.6, and 194.8 kDa); P1, anago as a positive control; P2, purified PA as a Lanepositive M, molecular control; 1, yoshikiri-zame weight marker; 2, ma-kasube（from ;bottom 3, kawa-yatsume to top:; 4,6.6, shira-uo 15.4,; 5, 20.7, hotei-uo 27.8,; 6, noro-genge 41.5, 56.8,; 7, kihada109.6,; 8, and kamasu-sawara 194.8 kDa）;; 9, hatahata P1, anago; 10, as a positive control; P2, purified PA as a positive control; 1,yoshikiri -zame; 2, ma-kasube; 3, kawa-yatsume; 4, shira-uo; shiro-saba-fugu; 11, manbou; 12, ma-kajika; 13, nore-sore; 14, nishin; 15, wakasagi; 16, shisyamo; 17, ayu; 18, komai; 19, suketou-dara; 20, hokke; 21, 5, hotei-uo; 6, noro-genge; 7, kihada; 8, kamasu-sawara; 9, hatahata; 10, shiro-saba-fugu; 11, manbou; 12, ma-kajika; hakkaku; 22, kurogashira-karei; and 23, karei. 13, nore-sore; 14, nishin; 15, wakasagi; 16, shisyamo; 17, ayu; 18, komai; 19, suketou-dara; 20, hokke; 21, hakkaku; 22, kurogashira-karei; and 23, karei.

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3 Parvalbumin Antigenicity and Allergenicity of 127 Species of Fish

kamasu-sawara, siro-saba-fugu, and manbou. at 0.81, was relatively high. Since the protein content was Red fish, such askihada and kamasu-sawara, showed calculated to be 32.5 mg/g, it was inferred that 54% of it a similar SDS-PAGE band pattern and had a large was PA, i.e., fish species with a high OD and low eluted amount of protein（14.8 and 12.3 mg/ml, respectively）. protein mass may have a low hypoallergenic effect due to Many of the muscles in red fish are slow muscles, and exposure to water. there are relatively few fast muscles, which is likely a Anago（sample 5）, hotaru-jyako（sample 104）, ebo-dai reason why the PA content was low. Although allergic （sample 107）, ma-haze（sample 80）, shita-birame（sam - reactions due to histamine poisoning are a concern with ple 119）, and chikame-kintoki（sample 112） showed a mackerel, there is a possibility that mackerel can be high level of specific activity, i.e., high IgG reactivity per 1 applied as a hypoallergenized food for PA-reactive allergic gram of protein, and the specific reactivity was much patients. higher than that of shiro-guchi, which has a high PA con- In addition, yoshikiri-zame, shira-uo, ma-kajika, and tent. Among the Salmonidae family, itou, salmon trout, shiro-saba-fugu were not reactive, and they had a large and yamame also showed a higher level of specific activ- amount of protein（7.2, 7.1, 5.8, and 7.2 mg/ml, respec- ity than shiro-guchi. These fish species, which appear to tively）. Although none of these fish species are widely have a higher PA content or higher reactivity than shiro- consumed, they may have clinical application in the treat- guchi, have a high PA to water-soluble protein ratio in ment of fish allergy caused by PA, and thus warrant fur- fish meat; thus, exposure to water is expected to have a ther studies. strong hypoallergenic effect on the fish meat. Quantification of PA in fresh fish meat using purified CONCLUSION PA and a hypoallergic approach for fish allergy patients Screening by ELISA is a qualitative assessment. For Among the 12 species of the Salmonidae family exam- further investigation of fish allergy, it is important to ined in our study, the antigenicity was the highest in itou purify and quantify PA. The PA content of four fish spe- （Japanese huchen）, and the lowest in chum salmon. The cies, as quantified by ELISA using purified PA as a stan- evaluation using the serum of a salmon PA-reactive dard, was 17.6 mg/g in shiro-guchi（ R 2＝0.97）, 6.1 mg/g patient showed that the IgE reactivity was significantly in shiro-zake （ R 2＝0.96）, 4.9 mg/g in toki-shirazu lower with shiro-zake（chum salmon） than with many of （R2＝0.97）, and 5.9 mg/g in shishamo（ R 2＝0.99）. the other salmonid fish species, indicating that allergic The patient with salmon allergy who provided AK-9 reactions are more probable with those salmonid fish spe- was able to eat shisyamo under the guidance of an allergy cies. Both salmon and trout are salmonid fish. Unfortu- specialist. The PA content of shishamo was 5.9 mg/g, nately, trout does not need to be labelled according to the which was lower than the 6.1 mg/g of PA in shiro-zake. current Japanese allergy labeling system even though it In addition, shiro-guchi, which is often used in processed appears to be more antigenic than salmon. Kihada（yel - fish products, was found to contain 17.6 mg/g of PA. lowfin tuna）,kamasu -sawara（wahoo）, shiro-saba-fugu Our research group has been trying to develop （green rough-backed puffer）, manbou（ocean sunfish）, chemical methods for enabling patients to achieve hypoal- shira-uo（ice fish）, and ma-kajika（great sculpin） are lergenicity; such methods include water-bleaching and considered to contain little PA. Yoshikiri-zame （blue extraction with potassium chloride（Kurata et al., 2017）, shark）, ma-kasube（mottled-skate）, and kawa-yatsume and enzymatic degradation（Kurata et al., 2018）. Even （Japanese lamprey） also have a low PA content, but they with such food treatment methods, it is still difficult to are cartilaginous fish, and are not usually consumed. Some completely eliminate the allergenicity of fish meat. In our fish was identified as having a low PA content and/or low previous paper, we reported that the PA content of allergenicity in our study, then it may be possible for mild shiro-guchi could be decreased down to five-fold of the allergy sufferers to eat those fish, or the fish could be detection limit when exposed to water（Kurata et al., applied in oral immune tolerance therapy as a hypoaller- 2017）. genic fish. In this study, the amount of water-soluble protein con- ACKNOWLEDGEMENTS tained in shiro-guchi was measured to be 6.5 mg/ml （sample 73）. In addition to PA, other water-soluble pro- This work was supported by the Japan Society for the teins were eluted in the extract used for ELISA. The OD, Promotion of Science（JSPS） KAKENHI（Grants-in-Aid

（153） 25 日本調理科学会誌（J. Cookery Sci. Jpn.） Vol. 52 No. 3（2019） for Scientific Research） Grant Number 18300258 for basic properties of salmon（Oncorhynchus nerka） between land- research on the construction and application of a database locked and anadromous species, Allergol. Int., 58, 295-299 Kurata, K,. Itoh, M., Matsumiya, M, Dobashi, A., Itagaki, Y., and for food allergens（fiscal years 2006-2008）. Kazuo, S.（2017）, Preparation of hypoallergenic Kamaboko: DISCLOSURES Removal of Parvalbumin and collagen from fish meat by water-bleaching, Mechanical grinding and extraction with The authors report no conflicts of interest in relation to potassium chloride, Nihon Chourikagaku Kaishi（ J. Cook- ery Sci. Jpn.）, 50, 141-150 this study. The funding agency（JSPS） was not involved Kurata, K,. Itoh, M., Matsumiya, M., Dobashi, A., Nakamura, A., in the design, collection, analysis and interpretation of Kondo, T., Osajima, K. and Itagaki, Y.（2018）, Reduction of data, writing of the paper, or the decision to submit the potential IgE reactivity in fish meat of Japanese anchovy by report. enzymatic degradation of parvalbumin and collagen, Nihon Chourikagaku Kaishi（ J. Cookery Sci. Jpn.）, 51, 205-216 Müntener, M., Kaser, L., Weber, J. and Berchtold, M. W.（1995）, REFERENCES Increase of skeletal muscle relaxation speed by direct injec- Arif, S. H. （2009）, A Ca2+ - binding protein with numerous tion of parvalbumin cDNA, Proc. Natl. Acad. Sci. U. S. A., roles and uses: parvalbumin in molecular biology and physi- 92, 6504-6508 ology, BioEssays, 31, 410-421 Rance, F., Grandmottet, X. and Grandjea, H.（2005）, Prevalence Ebisawa, M.（2018）, Rekishiteki Haikei to Gainenn no Henka, and main characteristics of schoolchildren diagnosed with “Syourei wo Toshite Manabu Nenndaibetu Syokumotu are- food allergies in France, Clin. Exp. Allergy, 35, 167-172 rugi no Subete”（in Japanese）, Nankoudou, Tokyo, p. 4 Sharp, M. F. and Lopata, A. L.（2014）, Fish Allergy: In review, Gerday, Ch.（1982）, Soluble calcium-binding proteins from fish Clin. Rev. Allergy Immunol., 46, 258-271 and invertebrate muscle, Mol. Physiol., 2, 63-87 Schiere, S. H.（2011）, Epideomiology of food allergy. J. Allergy Hajab, P. and Selamat, J.（2012）, A Contemporary review of Clin. Immunol., 127, 594-602 allergy, Clin. Rev. Allergy Immunol., 42, 365-385 Shimakura, K., Wataya, Y., and Shiomi, K.（2012）, Comparative Hilger, C., Thill, L., Grigioni, F., Lehners, C., Falagiani, P., analyses of allergens between landlocked and anadromous Ferrara, A., Romano, C., Stevens, W. and Hentges, F. species of masu salmon Oncorhynchus masou masou（in （2004）, IgE antibodies of fish allergic patients cross-react Japanese）, Shokuhin Eiseigaku Zasshi（ Food Hyg. Saf. with frog parvalbumin, Allergy, 59, 653-660 Sci.）, 53, 8-13 Itagaki, Y.（2011）, Food allergy and Fish（in Japanese）, Nihon Chourikagaku Kaishi（ J. Cookery Sci. Jpn.）, 44, 306-309 （Received Nov, 12, 2018 Accepted Feb, 24, 2019） Kondo, Y., Ahn, J., Komatsubara, R., Terada, A. Yasuda, T., Tsuge, I., and Urisu, A.（2009）, Comparison of allergenic

Appendix 1. Names of fishes used in this study and their Linnaean classifications, protein contents in the fish meats, and PA antigenicity against monoclonal anti-frog PA antibody.

I. Chondrichthyes Order of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 1 Carcharhiniformes Prionace glauca Blue shark Yoshikiri-zame 3.8 0.00 Megane-kasube, 2 Rajiforme pulchra Mottled skate 2.7 0.00 Ma-kasube Kawa-yatume, 3 Petromyzontiformes Lampetra japonica Japanese lamprey 7.3 0.00 Yatume-unagi

II. Anguilliformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 4 Anguillidae Anguilla japonica Japanese eel Unagi 6.5 1.19 5 Congridae Conger myriaster Common Japanese Conger Anago 5.0 1.93 6 - Juvenile conger Nore-sore 1.8 0.04 7 Muraenesocidae Muraenesox cinereus Daggertooth pike conger Hamo 3.9 0.32

26 （154） Parvalbumin Antigenicity and Allergenicity of 127 Species of Fish

III. Cluperiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 8 Clupeidae Clupea pallasii Pacific herring Nishin 3.5 0.06 9 Sardinops melanostictus Sardine Ma-iwashi 4.8 0.91 10 Sardinella zunasi Big-eye sardine Mamakari, Sappa 4.7 1.07 11 Konosirus punctatus Threadfin shad Konoshiro, Kohada 5.4 1.05 12 Engraulidae Engraulis japonica Japanese anchovy Katakuchi-iwashi 8.7 1.31 13 - Juvenile anchovy Shirasu 10.5 0.84

IV. Cyprinformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 14 Cyprinidae Cyprinus Carpio Carp Koi 8.1 1.34 15 Cobitidae Misgurnus anguillicaudatus Loach Dojyo 7.2 1.02

V. Salmoniformes Family of Scientific name of Protein Optical No Name in English Name in Japanese Linnaean classification Linnaean classification (mg/ml） density 16 Salmonidae Oncorhynchus nerka Sockeye salmon Beni-zake 9.0 0.65 17 Oncorhynchus gorbuscha Pink salmon Karafuto-masu 7.0 0.10 18 Oncorhynchus masou masou Cherry salmon Sakura-masu 8.8 0.34 19 Hucho perryi Japanese huchen Itou 6.1 1.45 20 Salvelinus leucomaenis pluvius Whitespotted char Iwana 5.9 0.59 21 Oncorhynchus nerka Kokanee Hime-masu 12.8 0.50 22 Oncorhynchus keta Chum salmon Shiro-zake 7.4 0.14 23 Oncorhynchus masou maso Land-locked trout Yamame 4.7 0.69 24 Oncorhynchus mykiss Salmon trout Salmon-trout 7.5 1.10

VI. Osemeriformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 25 Osmeridae Hypomesus nipponensis Japanese smelt Wakasagi 4.5 0.02 26 lanceolatus Shishamo smelt Shisyamo 4.3 0.07 27 Plecoglossus altivelis altivelis Japanese trout Ayu 5.6 0.03 28 Salangichthys microdon Icefish Shira-uo 7.1 0.00

VII. Gadiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 29 Gadidae Gadus chalcogrammus Alaska pollack Suketou-dara 5.5 0.01 30 Gadus macrocephalus Pacific cod Ma-dara 3.5 0.20 31 Eleginus gracilis Saffron cod Komai 4.3 0.00

VIII. Bericiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 32 Berycidae Beryx splendens Splendid alfonsino Kinme-dai 7.2 0.40

IX. Beloniformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 33 Scomberesocidae Cololabis saira Pacific saury Sanma 5.5 0.32 34 Exocoetidae Cypselurus agoo Japanese flyfish Tobi-uo 6.3 0.21 35 Hemiramphidae Hyporhamphus sajori Japanese halfbeak Sayori 8.9 1.15 36 Belonidae Strongylura anastomella Japanese needlefish Datsu 7.5 0.34

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X. Scorpeaniformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density Myoxocephalus Ma-kajika, 37 Cottidae Great sculpin 5.8 0.00 polyacanthocephalus Toge-kajika 38 Scorpaenidae Sebastiscus marmoratus Marbled rockfish Kasago 6.9 0.67 39 Sebastes schlegelii Black rockfish Kuro-soi 5.6 0.47 40 Sebastes kiyomatsui Kataboshi-red rockfish Kataboshi-akamebaru 5.6 0.48 41 Sebastes baramenuke Rose-rockfish Bara-menuke, 5.5 0.44 42 Sebastolobus macrochir Thornhead Kichiji, Kinki 3.8 0.56 43 Sebastes scythropus Sullen rockfish Ukeguchi-mebaru 5.0 0.36 44 Platycephalidae Platycephalus sp.2 Bartail flathead Ma-gochi, Kochi 8.2 0.26 45 Triglidae Chelidonichthys spinosus Bluefin searobin Hou-bou 7.7 0.57 46 Hexagrammidae Hexagrammos otakii Green ling Ainame 4.7 0.26 47 Pleurogrammus azonus Atka mackerel Hokke 5.0 0.03 48 Agonidae Podothecus sachi Sailfin poacher Hakkaku, Tokubire 4.4 0.06 49 Cyclopteridae Aptocyclus ventricosus Smooth lumpsucker Hotei-uo, Gokko 2.7 0.00

XI. Mugiliformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 50 Mugilidae Mugil cephalus cephalus Flathead gray mullet Bora 7.4 0.31

XII. Gasteristeiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 51 Fistulariidae Fistularia petimba Smooth flutemouth Aka-yagara 9.5 0.20

XIII. Siluriformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 52 Siluridae Silurus asotus Japanese catfish Namazu 7.3 0.66

XIV. Aulopiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 53 Synodontidae Saurida macrolepis Brushtooth lizardfish Eso, Ma-eso 4.4 0.85 54 Chlorophthalmidae Chlorophthalmus albatrossis Bigeyed greeneye Mehikari, Aome-eso 5.4 0.08

XV. Zeniformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 55 Zeniontidae Zeus faber John dory Matou-dai 13.2 1.08

XVI. Perciformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 56 Lateolabracidae Lateolabrax japonicus Japanese seaperch Suzuki 5.6 0.17 57 Zoarcidae Bothrocara hollandi Porous-head eelpout Noro-genge 2.0 0.00 58 Rachycentridae Rachycentron Canadum Cobia, Black kingfish Sugi 10.3 0.12 59 Kyphosidae Labracoglossa argentiventris Yellowstriped butterfish Takabe 7.2 0.98 60 Trachurus japonicus Japanese horse-mackarel Ma-aji 4.1 0.81 61 Seriolina nigrofasciata Blackbanded trevally Ai-buri 8.6 0.03 62 dumerili Greater Kanpachi 8.9 0.42 63 Decapterus muroadsi Amberstripe scad Muro-aji 7.1 0.64 64 Alectis ciliaris Giliated threadfish Itohiki-aji 6.5 0.67

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65 Seriola quinqueradiata Buri 10.2 0.46 66 Pseudocaranx dentex Shima-aji 9.9 0.05 67 Caranx sexfasciatus Crevalle jack Gingame-aji 6.8 0.94 68 Elagatis bipinnulata Rainbow runner Tsumu-buri 7.1 0.55 69 Seriola lalandi Hiramasa 7.8 0.10 70 Uraspis helvola Whitetongue jack Oki-aji 6.4 0.43 71 Sparidae Pagrus major Red sea-bream Ma-dai 9.3 0.41 72 Acanthopagrus schlegelii Blackhead sea-bream Chinu, Kuro-dai 7.6 1.36 73 Sciaenidae Pennahia argentata White croaker Shiro-guchi 6.5 0.81 74 Coryphaenidae Coryphaena hippurus Common dolphinfish Shiira, Mahimahi 6.4 0.04 75 Sillaginidae Sillago japonica Japanese whiting Shiro-gisu 6.3 0.72 76 Scombropidae Scombrops gilberti Japanese bluefish Kuro-mutsu 6.0 1.31 77 Haemulidae Parapristipoma trilineatum Chicken grunt Isaki 7.4 0.55 78 Plectorhinchus cinctus Spotted grunt Koshou-dai 5.1 0.64 79 Diagramma picta Painted sweetlip Koro-dai 5.9 0.32 80 Gobiidae Acanthogobius avimanus Yellowfin goby Ma-haze 5.5 1.51 81 Serranidae Epinephelus bruneus Longtooth grouper Kue, Ara 5.0 0.65 Red-spotted rock-cod, 82 Plectropomus leoparadus Suji-ara 6.0 0.64 Blue spotted grouper Sea-bass, Grouper, 83 Niphon spinosus Ara 7.0 0.99 Rock-cod 84 Labridae Choerodon azurio Tuskfish Ira 5.2 0.04 85 Scomber japonicus Chub mackerel Ma-saba 7.9 0.04 86 Thunnus orientalis Pacific bluefin tuna Kuro-maguro 8.8 0.27 Striped tuna, Skipjack 87 Katsuwonus pelamis Katsuo 10.2 0.63 tuna, Oceanic Japanese Spanish 88 Scomberomorus niphonius Sawara 6.3 0.74 mackerel 89 Thunnus albacares Kihada 14.8 0.00 90 Acanthocybium solandri Wahoo Kamasu-sawara 12.3 0.01 91 Euthynnus afnis Wavyback skipjack Suma 9.4 0.40 Souda-katsuo, 92 Auxis thazard thazard Frigate tuna 9.2 0.16 Hira-souda 93 Sphyraenidae Sphyraena pinguis Barracuda Hon-kamasu 5.6 1.07 94 Girellidae Girella punctata Greeenfish Mejina 6.3 1.35 95 Scaridae Chlorurus microrhinos Blunt-headed parrotfish Nanyou-budai 5.6 0.85 96 Scarus rubroviolaceus Parrotfish Naga-budai 4.9 0.40 97 Trichiuridae Trichiurus japonicus Largehead hairtail Tachi-uo 4.8 0.48 98 Japanese sandfish Hatahata 2.1 0.00 99 Oplegnathus fasciatus Barred knifejaw Ishi-dai 6.5 1.04 Oplegnathidae 100 Oplegnathus punctatus Parrot-bass Ishigaki-dai 3.0 0.01 101 Platycephalidae Suggrundus meerdervoortii Big-eyed flat-head Megochi 6.7 0.95 102 Nemipteridae Nemipterus virgatus Golden threadfin-bream Itoyori, Itoyori-dai 5.3 0.52 Ama-dai, 103 Branchiostegidae Branchiostegus japonicus Horsehead tilefish 6.1 0.57 Aka-ama-dai, Guji 104 Acropomatidae Acropoma japonicum Lanternbelly Hotaru-jyako 4.8 1.64 105 Doederleinia berycoides Blackthroat seaperch Aka-mutsu, Nodoguro 6.2 0.33 106 Kyphosidae Kyphosus vaigiensis Waigeu drummer Isu-zumi 7.5 1.03 107 Centrolophidae Psenopsis anomala Wart parch Ebo-dai, Ibo-dai 5.4 1.69 108 Hyperoglyphe japonica Japanese butterfish Me-dai 6.3 0.09 109 Caesionidae Pterocaesio diagramma Black-tip fusilier Takasago 8.0 0.13

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110 Lethrinidae Gymnocranius euanus Japanese large-eye bream Shiro-dai 9.9 1.38 111 Stromateidae Pampus punctatissimus Harvest fish Mana-gatsuo 4.4 0.07 112 japonicus Longfinned bullseye Chikame-kintoki 6.2 1.53 113 Pholidae Pholis nebulosa Bllenny Ginnpo 5.1 0.71 114 Gempylidae Promethichthys prometheus Snake-mackerel Kurobisi-kamasu 5.2 0.03

XVII. Pleuronectiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density 115 Paralichthys olivaceus Japanese flounder Hirame 7.6 0.50 116 Righteye flounder Mako-garei, Karei 6.6 0.00 yokohamae 117 Cleisthenes pinetorum Souhachi-flounder Souhachi-garei 6.3 0.06 Kurogashira-garei, 118 Pseudopleuronectes schrenki Cresthead flounder 5.6 0.00 Kuro-garei Shita-birame, 119 Cynoglossidae Paraplagusia japonica Tonguefish 6.0 1.62 kuro-ushinoshita

XVIII. Tetraodonitiformes Family of Linnaean Scientific name of Protein Optical No Name in English Name in Japanese classification Linnaean classification (mg/ml） density Green rough-backed 120 Tetraodontidae Lagocephalus wheeleri Shiro-saba-fugu 7.2 0.00 puffer 121 Monacanthidae Stephanolepis cirrhifer Thread-sail filefish Kawa-hagi 7.0 0.77 122 Thamnaconus modestus Filefish Umadura-hagi 5.9 1.07 123 Molidae Mola sp.B Ocean sunfish Manbou 2.6 0.00 124 Lophiidae Lophiomus setigerus Goosefish Ankou 2.0 0.07

抗カエル PA 抗体および患者血清を用いた ELISA による 127 魚種の パルブアルブミンのアレルゲン性評価

倉田香織*§ 土 橋 朗* 栗 原 和 幸** 板 垣 康 治***

和文抄録 魚肉に含まれる主要なアレルゲンの一つであるパルブアルブミン（PA）の含有量は魚種により異なると考えられる。本 研究では，抗カエル PA 抗体および患者血清を用いた ELISA により，サケ科 12 魚種の中で，シロザケが最も反応性が低 いことを明らかにした。陸封型サケの抗原性は陸海型サケのそれよりも高く，特定原材料のアレルギー表示において陸封 型のトラウトが表記されていない問題を明らかにした。124 魚種の抗原性スクリーニングの結果，アナゴが最も高い抗原 性を示した。硬骨魚鋼では，サバフグやシラウオが低かった。軟骨魚鋼であるメガネカスベ，ヨシキリザメ，カワヤツメ の抗原性も低かった。精製 PA を用いた定量の結果，シログチ 17.6 mg/g，シロサケ 6.1 mg/g，トキシラズ 4.9mg/g， シシャモ 5.9 mg/g であった。

キーワード：食物アレルギー，魚類アレルギー，パルブアルブミン，軟骨魚類，ELISA

* 東京薬科大学 ** 神奈川県立こども医療センター *** 神奈川県衛生研究所 現職：北海道文教大学 § 責任著者連絡先 東京薬科大学情報教育研究センター 〒 192-0392 東京都八王子市堀の内 1432-1 TEL 042（676）3095 FAX 042（670）7067 E-mail：[email protected]

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