Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Development of an Indirect Enzyme-Linked Immunosorbent assay for Rapid Detection of Adulteration of Food Allergen Mackerel in Processed Marine Foods

수산가공식품 중 알레르겐 고등어 혼입여부 신속 검출을 위한 간접효소면역분석법의 개발

  • Lee, Jeong-Eun (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • Kim, Ah-Yoon (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • Kim, Sol-A (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • Kim, Hyo-In (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • Park, Ji-Hye (Division of Applied Life Science, Graduate School, Gyeongsang National University) ;
  • Shim, Won-Bo (Institute of Agriculture and Life Science, Gyeongsang National University)
  • 이정은 (경상대학교 응용생명과학부) ;
  • 김아윤 (경상대학교 응용생명과학부) ;
  • 김솔아 (경상대학교 응용생명과학부) ;
  • 김효인 (경상대학교 응용생명과학부) ;
  • 박지혜 (경상대학교 응용생명과학부) ;
  • 심원보 (경상대학교 농업생명과학연구원)
  • Received : 2018.04.10
  • Accepted : 2018.05.28
  • Published : 2018.06.30
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Abstract

The purpose of this study was to develop an indirect enzyme-linked immunosorbent assay (indirect ELISA) based on a monoclonal antibody (MAb) that is specific to mackerel thermal stable-soluble protein (TSSP), that can be used for the rapid detection of mackerel in processed marine foods. Among the four MAbs (3A5-1, 2, 9, and 12) developed in previous studies, the 3A5-2 MAb that showed high specificity and sensitivity were selected and used to develop the indirect ELISA method. The detection range of the indirect ELISA was 0.02%-0.001% and the detection limit of 0.001% was shown. No cross-reaction to other marine products and food ingredients was observed by the indirect ELISA. Processed marine foods containing mackerel with ${\geq}0.3$ O.D. value at 405 nm were estimated as positive samples by the indirect ELISA. Therefore, the indirect ELISA can be used as a rapid and sensitive method to identify mackerel authenticity and adulteration in processed marine foods.

본 연구에서는 수산가공품 중 고등어 어육을 신속하게 검출하기 위하여 고등어 어육 중 열 안정-수용성 단백질에 특이한 단클론성 항체(3A5-2)를 이용하여 indirect ELISA 법을 개발하였다. Indirect ELISA을 개발하기에 앞서 먼저 시료 전처리는 이전 연구의 결과를 바탕으로 진행되었다. 이전 연구에서는 3A5-2 항체가 37 kDa 부근의 열 안정-수용성 단백질과 반응하였고, 0.05 M carbonate buffer로 추출하였을 때 흡광도가 가장 두드러지게 증가한 것을 확인하였다. 따라서 indirect ELISA의 시료 전처리는 0.05 M carbonate buffer를 이용한 열처리 추출법으로 추출한 후 0.05 M PBS로 희석하는 것으로 확립하였고, indirect ELISA 법을 최적화하였다. 개발된 indirect ELISA법에 실험실에서 임의로 열처리한 고등어 샘플을 적용한 결과 indirect ELISA법은 0.001% (0% 흡광도 표준편차 양의 값: $0.0003{\times}3$)의 검출한계를 확인하였으며, 꽁치 중 고등어는 0.002%(0% 흡광도 표준편차 양의 값: $0.0006{\times}3$)까지 검출이 가능하였다. 또한 조리($100^{\circ}C$, 30분)와 멸균($121^{\circ}C$, 30분) 처리된 고등어의 검출이 가능한 것으로 확인되었고, 멸균된 제품에서도 고등어에 특이적으로 반응하여 고등어의 혼입여부 판별도 가능한 것으로 판단되었다. 시판되는 고등어 가공품에 대해서는 양성결과를 나타내었고 다른 수산물에서는 음성으로 판단되어 개발된 분석법은 가공품에 혼입될 수 있는 알레르겐인 고등어를 보다 신속하고 민감하게 분석할 수 있고, 점차 가격이 증가하고 있는 고등어의 혼입여부를 확인할 수 있는 분석 도구로서 활용이 가능할 것으로 판단된다.

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References

  1. Lee H.K., Song W.J., Ha T.U.: A single or combined effects of IL-4 and other various cytokines on IgE production of human tonsillar mononuclear cells. Korean J. Immunol., 17, 193-201 (1995).
  2. Okunuki H, Teshima R, Sakushima J, Akiyama H, Goda Y, Toyoda M, Sawada JI. Induction of active systemic anaphylaxis by oral sensitization with ovalbumin in mast-cell-deficient mice. Immunol. Lett., 74, 233-237 (2000). https://doi.org/10.1016/S0165-2478(00)00264-9
  3. Broide DH. Molecular and cellular mechanisms of allergic disease. J. Allergy Clin. Immun., 108, S65-S71 (2001). https://doi.org/10.1067/mai.2001.116436
  4. Lim, K.M., Park S.M., Shim, B,S., Kim, S.K., Choi, S.L., Ahn K.S.: Effect of bulhwangeumjeonggi-san on cytokine levels of mouse th1/th2 cells and anti-allergic activity in ovalbumin-sensitized allergic inflammation model. Korean J. Oriental Physiology&Patholog., 20(6), 1467-1476 (2006).
  5. The Korean academy of asthma allergy clinical immunology. Asthma and Allergic Diseases. Yeomungag Press, 445-469 (2012).
  6. Lee, J.E., Kim, J.S., Shim, W.B.:Production of A monoclonal antibody (mab) against a thermal stable-soluble protein in mackerel and confirmation of the properties for the MAb. J. Food Hyg. Saf., 32, 1-7 (2017). https://doi.org/10.13103/JFHS.2017.32.1.1
  7. George, C.W., Susott, R.A.: Effects of ammonium phosphate and sulphate on the pyrolysis and combustion of cellulose. USDA Forest Service INT-90 (1971).
  8. Loiseau, O., Cottin, J., Robert, R., Tronchin, G., Mahaza, C., Senet. J.M.: Development and characterization of monoclonal antibodies specific for the genus Listeria. Immunol. Med. Microbiol., 11, 219-230 (1995). https://doi.org/10.1111/j.1574-695X.1995.tb00120.x
  9. Towbin, H., Staehelin, T., Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci., 76, 4350-4354 (1979). https://doi.org/10.1073/pnas.76.9.4350
  10. Faeste, C.K, Plassen C. Quantitative sandwich ELISA for the determination of fish in foods. J. Immunol., 329, 45-55 (2008).
  11. Sletten G, Van Do T, Lindvik H, Egaas E, Florvaag E. Effects of industrial processing on the immunogenicity of commonly ingested fish species. Int. Arch. Allergy Imm., 151, 223-236 (2010). https://doi.org/10.1159/000242360
  12. Taylor SL, Nordlee JA, Niemann LM, Lambrecht DM. Allergen immunoassays-considerations for use of naturally incurred standards. Anal. Bioanal. Chem., 395, 83-92 (2009). https://doi.org/10.1007/s00216-009-2944-0
  13. Son, D.H. and Kim, M.H.: Development of sandwich ELISA for the detection of mackerel in processed foods. Korean J. Food Sci. Technol., 44(1), 1-7 (2012). https://doi.org/10.9721/KJFST.2012.44.1.001
  14. Chen, F.C., Hsieh, Y-H.P.: Porcine troponin I: a thermostable species marker protein. Meat Sci., 61, 55-60 (2002). https://doi.org/10.1016/S0309-1740(01)00162-0
  15. Nhari, R., Hafidz, R.M., Hamid, M., Rasli, N.M., Omar, A.R., Sheikha, E., Mustafa, S.: Monoclonal antibodies specific to heat-treated porcine blood. J. Sci. Food Agric., 96, 2524-2531 (2015).
  16. Chen, F.C., Hsieh, Y-H.P., Bridgman, R.C.: Monoclonal antibodies to porcine thermal-stable muscle protein for detection of pork in raw and cooked meats. J. Food Sci., 63, 201205 (1998).
  17. Marion, M., Bradford: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254 (1976). https://doi.org/10.1016/0003-2697(76)90527-3
  18. Asensio, L., Gonzlez, I., Garca T., Martn, R. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control, 19, 1-8 (2008). https://doi.org/10.1016/j.foodcont.2007.02.010
  19. Lee, J.H., Song, K. Y., Hyeon, J.Y., Hwang, I.G., Kwak, H.S., Han, J. A., Chung, Y. H., Seo, K. H.: Comparison of standard culture method and real-time pcr assay for detection of Staphylococcus aureus in processed and unprocessed foods. Korean J. Food Sci. An., 3, 410-418 (2010).
  20. Hwang, E.S., Lee, T.U. Jung, D.Y., Cho, H.S.; Development of loop-mediated isothermal amplification method for the rapid and sensitive detection of bovine tuberculosis in Korea native cattle. Korean J. Veterinary Service., 4, 333-339 (2011).
  21. MFDS (Ministry of Food and Drug Safety): Ministry of Food and Drug Safety Notice 2017-15, Available from https://www.mfds.go.kr/index.do?mid=686&pageNo=1&seq=11721&sitecode/ Accessed Mar. 08 (2017).
  22. KOSIS (Korean Statistical Information Service): A survey of fisheries production trends, Available from http://kosis.kr/statHtml/statHtml.do?orgId=101&tblId=DT_1W0002vw_cd=MT_ZTITLE&list_id=F37&seqNo=&lang_mode=ko&language=kor&obj_var_id=&itm_id=&conn_path=MT_ZTITLE/ Accessed Aug. 25 (2017).
  23. KCA (Korea Consumer Agency): Need to improve labeling system for food allergens, Available from http://www.kca.go.kr/brd/m_32/view.do?seq=2395&multi_itm_seq=0/ Accessed Mar. 14 (2018).
  24. NSC (Norwegian Seafood Council): 2017 Research on marine product consumption, Available from https://norge.co.kr/contentassets/f77cd3f4658e475b9f22b1024eb6e90a/nsc_seafood-study-2017/ Accessed Nov. 23 (2017).