280 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.0,,2*,20 (,**1) 34 ELISA * * * * ** Determination of Crustacean Allergen in Food Products b

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1 280 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.0,,2*,20 (,**1) 34 ELISA * * * ** ** * ** Determination of Crustacean Allergen in Food Products by Sandwich ELISA Yusuke Shibahara, Michihiro Oka, Kei Tominaga, Toshihiro Ii, Mamoru Umeda, Noriko Uneo*, Akihisa Abe*, Eiji Ohashi*, Hideki Ushio** and Kazuo Shiomi** Research Division, Nissui Pharmaceutical Co., Ltd., +*1/, Hokunanmoro Yuki-shi, Ibaraki -*1 **-0 * Food Safety Research Center, Nippon Suisan Kaisha, Ltd., //3-0 Kitano-Machi, Hachioji-shi, Tokyo +3, *3*0 ** Faculty of Marine Science, Tokyo University of Marine Science and Technology,. / 1 Konan, Minato-ku, Tokyo +* Crustaceans are one of the most significant sources of food allergies, and tropomyosin is their major allergen. To detect crustacean allergens in food products, we have developed a sandwich ELISA using a crustacean tropomyosin-specific monoclonal antibody using black tiger prawn tropomyosin as an immunogen. Our ELISA showed 2,*, reactivity (based on reactivity to black tiger prawn tropomyosin) to tropomyosins from crustaceans including prawns, lobster, crab, hermit crab, and krill, while no substantial reactivity ( *.+ ) to those from mollusks, including squid, octopus, and shellfish, was observed. Furthermore, no positive ELISA results were obtained with any crustacean-free foods. Repeatability coe$cients of variation (CVs) were *, and the detection limit based on a standard crustacean protein was *.+0 ppm, which qualified for the sensitivity requirements of the current food labeling system. The ELISA could detect crustacean tropomyosin, even heat-denatured tropomyosin, without being a#ected by food matrices. These results indicated that this ELISA is highly specific to crustacean tropomyosin, and will be useful as a method for detecting crustacean allergens in processed food products. (Received Jan..,,**1 ; Accepted Mar. +3,,**1) Keywords : food allergy, crustaceans, tropomyosin, ELISA, monoclonal antibody : ELISA +.. / +,*, -*1 **-0 +*1/, * 3, *3*0 //3 0 ** * / 1 Corresponding author y-shibahara@yki.nissui-pharm.jp EU

2 35 : kda / , Enzyme-Linked Immunosorbent Assay ; ELISA ;, -, GREER Laboratories, 0 1 Greaser Gergely +- Motoyama 0 HPLC +- SDS SDS-PAGE SDS-PAGE Xcell Sure Lock Mini-Cell Invitrogen NuPAGE., Bis-Tris Gels Invitrogen - g /,, SDS +* See- Blue Plus, Pre-Stained Standard Invitrogen Coomassie Brilliant Blue R-,/* - -** g, ; Freund s Adjuvant, Complete, Sigma *, HiTrap Protein G HP Amersham Biosciences +. BALB c +** g, ; TiterMax Gold, Sigma * +*,* -* -/ P-X0-Ag2 +/,++,, + ELISA, g ml +** L. / +** g ml / / + /* L +, g ml IgG Biosource +** L +

3 282 /. 0,**1 0 36, mmol L.../ mmol L +** L -* *.2 /* L.3* nm, 0/* nm, SDS *./ +** /,+ 0*, g ml + ELISA, HiTrap Protein G HP Amersham Biosciences. *.+ g *./ SDS,,, +* L ml Halt Protease Inhibitor Cocktail EDTA Solution Halt Protease Inhibitor Cocktail Kit, Pierce ph 1.. *.*+ mol L Phosphate Bu#erd Saline ; PBS,* ml / +* *** g -* *.2 m DISMIC-,/cs, Advantec +** +*,-D Quant kit Amersham Biosciences PBS ph 1.. +* *., PBS ph 1.. / + g +3 ml,,* - *** g,*,* 0 ELISA,/ g ml *.+ mol L ph 0.* -,-,/,/ *./ mol L : * *.12 +./ ,/ +,./,/ /* ng ml +** L + / +** L + / +** L,* +** L./* nm, 0/* nm :. 2,*,*.** *.12 /* ng ml *.-+,* ppm + + +, A B C D + A,1, SDS SDS SDS SDS +. ELISA IgG + + *.12 /* ng ml, -0 *.. *.2 +.0,.. -.,..* ng ml * ISO IUPAC +0 *.. ng ml

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6 39 : 285. n -, +* +0 +4, -4* +4/ 224, 3*4, +*,4/ -4., *.4/ ** * ,41,41 + ppm ELISA 22.-** , 0+ /0 /2 0 1 IgE, 0 1 IgE / ELISA -, +* +0 ppm *../ 22.,*,./ 3-.1*..- +*. 0 * ppm 0* -* ** -*,+ +* ELISA - 0* -* ** -* ,+ +* 2*.* ELISA ELISA 2,*, *.+ *.+0 ppm ppm +*

7 286 /. 0,** /, 0/ 1-,**,, + pp.,.+,..,**, - Reese, G., Ayuso, R. and Lehrer, S.B., Tropomyosin : an invertebrate pan-allergen. Int. Arch. Allergy Immunol., ++3,,.1,/2 (+333).. Lehrer, S.B. and Ayuso, R., Seafood allergy and allergens : a review. Mar. Biotechnol., /, (,**-). / Ho#man, D.R., Day, E.D. Jr. and Miller, J.S., The major heat stable allergen of shrimp. Ann.Allergy,.1, +1,, (+32+). 0 Motoyama, K., Suma, Y., Ishizaki, S., Nagashima, Y. and Shiomi, K., Molecular cloning of tropomyosins identified as allergens in six species of crustaceans. J. Agric. Food Chem., //, 32/ 33+ (,**1). 1 Leung, P.S.C., Chow, W.K., Du#ey, S., Kwan, H.S., Gershwin, M.E. and Chu, K.H., IgE reactivity against a cross-reactive allergen in Crustacea and Mollusca : evidence for tropomyosin as the common allergen. J. Allergy Clin. Immunol., 32, 3/. 30+ (+330). 2 Santos, A.B.R., Chapman, M.D., Aalberse, R.C., Vailes, L. D., Ferriani, V.P.L., Oliver, C., Rizzo, M.C., Naspitz, C.K. and Arruda, L.K., Cockroach allergens and asthma in Brazil : identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J. Allergy Clin. Immunol., +*., -,3 --1 (+333). 3 Ayuso, R. and Lehrer, S.B., Identification of continuous, allergenic regions of the major shrimp allergen Pen a + (tropomyosin). Int. Arch. Allergy Immunol., +,1,,1-1 (,**,). +* Ayuso, R., Reese, G., Leong-Kee, S., Plante, M. and Lehrer, S.B., Molecular basis of arthropod cross-reactivity : IgEbinding cross-reactive epitopes of shrimp, house dust mite and cockroach tropomyosins. Int. Arch. Allergy Immunol., +,3, -2.2 (,**,). ++ pp. 22+-,**- +, Lu, Y., Ohshima, T., Ushio, H., Hamada Y. and Shiomi, K., Immunological characteristics of monoclonal antibodies against shellfish major allergen tropomyosin. Food Chem., +**, +*3-*33 (,**1). +- Greaser, M.L. and Gergely, J., Reconstitution of troponin activity from three protein components. J. Biol. Chem.,,.0,.,,0.,-- (+31+). +., pp. 01,1 32, +/ Köhler, G. and Milstein, C., Continuous cuitures of fused cells secreting antibody of predefined specificity. Nature,,/0,.3/.31 (+31/). +0 ICH Haromonized Tripartite Guideline, Varidation of analytical procedures : Methodology (+330)

320 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.1, -,* -,/ (,**1) 8 * * * * E#ect of Superheated Steam Treatment on the Preservation an

320 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /., No.1, -,* -,/ (,**1) 8 * ** *** * ** *** E#ect of Superheated Steam Treatment on the Preservation and Sensory Characteristics of Buckwheat Noodles Kazuhiro