Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Competitive ELISA for the Measurement of Glycoprotein Purified from Acanthopanx senticosus

가시오가피로부터 분리한 단백 다당물질의 경쟁적 ELISA법에 의한 분석

  • Ha, Eun-Suk (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Hwang, Soo-Hyun (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Shin, Kwang-Soon (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Yu, Kwang-Won (Department of Kimchi and Food Seience, National College of Science and Technology) ;
  • Lee, Keyung-Ho (Kolon Central Research Park) ;
  • Choi, Joo-Sun (Division of Metabolic Disease, Department of Biomedical Science, National Institute of Health) ;
  • Park, Woo-Mun (Research Team, GOOFOO Inc) ;
  • Yoon, Taek-Joon (Department of Food Science and Biotechnology, Kyonggi University)
  • 하은숙 (경기대학교 식품생물공학과) ;
  • 황수현 (경기대학교 식품생물공학과) ;
  • 신광순 (경기대학교 식품생물공학과) ;
  • 유광원 (청주대학교 김치식품과학과) ;
  • 이경호 (코오롱 중앙연구소) ;
  • 최주선 (국립보건원 대사영양질환과) ;
  • 박우문 ((주)구푸) ;
  • 윤택준 (경기대학교 식품생물공학과)
  • Published : 2003.12.01
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Abstract

This study was carried out to establish a quantitative analysis method of separating immuno-activating substance (EN-SP) from Acanthopanax senticosus (A. senticosus) by competitive direct ELISA. Mouse antiserum (anti-EN-SP) against EN-SP was generated by immunization (s.c.) of EN-SP purified from A. senticosus as an immunogen. The titer of anti-EN-SP was about 1 : 400, and the optimal dilution of EN-SP-HRP conjugate was 1 : 1,000. When the standard curve was constructed by ELISA, its sensitivity was about $0.2{\mu}g/mL$. The coefficient variation of intra- and inter-assay were $6.13{\sim}8.81%$ and $6.73{\sim}8.60%$, respectively. According to the standard curve, the concentration of EN-SP in various senticosus extracts was found to be only $59.85\;{\mu}g$ in 10mg of extract from the bark of A. senticosus. Similarly, the immunostimulating activity to produce $TNF-{\alpha}$ or IL-12 among the various extracts of Acanthopanax was shown to be correlated with the content of EN-SP. These results demonstrated that competitive ELISA was a convenient, fast, reproducible, and accurate method for the determination of EN-SP as an immunologically active standard substance in extract of A. senticosus.

가시오가피 냉침 추출물에서 면역자극활성을 가지는 지표물질을 분리하고자, gel chromatography를 수행하여 비장세포에 대한 증식활성을 가지는 EN-SP을 분리하였고, 생화학적 분석 결과 단백 다당 물질임을 확인하였다. 가시오가피 추출물 내에서 지표물질로서 EN-SP 성분을 정량하기 위하여 경쟁적 ELISA법을 개발하여 다음과 같은 결과를 얻었다. 경쟁적 ELISA법을 이용하여 EN-SP-HRP conjugate의 희석농도를 1000배로 결정하였으며, EN-SP의 표준곡선을 작성한 결과 검출범위는 $0.2{\sim}20\;{\mu}g/mL$으로서 측정감도는 $0.2\;{\mu}g/mL$이었다. 이러한 표준곡선의 정당성 평가를 위하여 정량된 EN-SP를 통한 재현성 실험에서 $5\;{\mu}g/mL$ 이하의 농도에서 intra assay 경우 C.V. 값은 $6.13{\sim}8.81%$, bias는 평균 -3.7%를 보였으며, inter assay에서도 C.V. 값과 bias 모두에서 10%이내로 비교적 우수한 재현성을 보였다. 작성된 표준곡선을 바탕으로 유사물질인 오가피의 $4^{\circ}C$ 추출물들의 EN-SP의 함유량을 조사한 결과 EN-SP 성분은 주로 가시오가피 외피에서만 높은 함유량을 보였다. 동일한 추출물의 macrophage 자극에 의한 cytokine 유도활성 실험결과, EN-SP 성분을 함유하는 가시오가피의 수피 추출물만 IL-12 및 $TNF-{\alpha}$를 생산하는 면역자극활성을 나타냄으로서 EN-SP 성분은 가시오가피에서 면역자극활성을 나타내는 지표물질로의 가능성이 제시되었다.

Keywords

References

  1. Ohnishi, Y, Fujii, H., Hayakawa, Y., Sakukawa, R., Yamarura, T., Sakamoto, T., Tsukada, K, Fujimaki, M., Nunome, S., Komatsu, Y. and Saiki, I. Oral administration of kampo (Japanese herbal) medicine Juzen-taiho-to inhibits liver metastasis of colon 26-L5 carcinoma cells. Japan J. Cancer Res. 89: 206-213 (1998) https://doi.org/10.1111/j.1349-7006.1998.tb00550.x
  2. Parhami-Seren, B., Bell, C., Margolies, M.N., and Haupert, G.T. Monoclonal antibodies that distinguish between two related digitals glycosides, Ouabain an Digoxin. J. Immunol. 163: 4360-4366 (1999)
  3. Yi, J.M., Kim, M.S., Seo, S.W., Lee, K.N., Yook, C.S. and Kim, H.M. Acanthopanax senticosus root inhibits mast cell-dependent anaphylaxis. Clin. Chim. Acta 312: 163-168 (2001) https://doi.org/10.1016/S0009-8981(01)00613-1
  4. Jeong, H.J., Koo, H.N., Myung, N.I., Shin, M.K., Kim, J.W., Kim, D.K., Kim, K.S., Kim, H.M. and Lee., Y.M. Inhibitory effects of mast cell-mediated allergic reactions by cell cultured Siberian ginseng. Immunopharmacol. Immunotoxicol. 23: 107-117 (2001) https://doi.org/10.1081/IPH-100102572
  5. Yoon, T.J., Lee, S.W., Shin, K.S., Choi, W.H., Hwang, S.H., Seo, S.H., Kim, S.H. and Park, W.M. Effect of hot water extract from Acanthopanax senticosus on systemic anaphylaxis. Korean J. Food Sci. Technol. 34: 518-523 (2002)
  6. Park S.Y., Chang, S.Y., Yook, C.S. and Nohara, T. New 3,4-secolupane-type triterpene glycosides from Acanthopanax senticosus forma inermis. J. Nat. Prod. 63: 1630-1633 (2000) https://doi.org/10.1021/np000277c
  7. Bae, E.A., Yook, C.S., Oh, O.J., Chang, S.Y, Nohara, T. and Kim, D.H. Metabolism of chiisanoside from Acanthopanax divaricatus var. albeofructus by human intestinal bacteria and its relation to some biological activities. BioI. Pharm. Bull. 24: 582-585 (2001) https://doi.org/10.1248/bpb.24.582
  8. Davydov, M. and Krikorian, A.D. Eleutherococcus senticosus Maxim. (Araliaceae) as an adaptogen: a closer look. J. Ethnopharmacol. 72: 345-393 (2000) https://doi.org/10.1016/S0378-8741(00)00181-1
  9. Dowling, E.A., Redondo, D.R., Branch, J.D., Jones, S., McNabb, G. and Williams, M.H. Effect of Eleutherococcus senticosus on submaximal and maximal exercise performance. Med. Sci. Sports Exerc. 28: 482-489 (1996) https://doi.org/10.1097/00005768-199604000-00013
  10. Gaffney, B.T., Hugel, H.M. and Rich, P.A. Panax ginseng and Eleutherococcus senticosus may exaggerate an already existing biphasic response to stress via inhibition of enzymes which limit the binding of stress hormones to their receptors. Med. Hypotheses 56: 567-572 (2001) https://doi.org/10.1054/mehy.2000.1163
  11. Lin, C.C. and Huang, P.C. Antioxidant and hepatoprotective effects of Acathopanax senticosus. Phytother. Res. 14: 489-494 (2000) https://doi.org/10.1002/1099-1573(200011)14:7<489::AID-PTR656>3.0.CO;2-G
  12. Umeyama, A., Shoji, N., Takei, M., Endo, K. and Arihara, S. Ciwujianosides DI and CI : powerful inhibitors of histamine release induced by anti-immunoglobulin E from rat peritoneal mast cells. J. Pharm. Sci. 81: 661-662 (1992) https://doi.org/10.1002/jps.2600810714
  13. Hacker, B. and Medon, P. Cytotoxic effects of E. senticosus aqueous extract against Ll210 leukemia cells. J. Pharm. Sci. 73:270272 (1984) https://doi.org/10.1002/jps.2600730235
  14. Hibasami, H., Fujikawa, T., Takeda, H., Nishibe, S., Satoh, T., Fujisawa, T. and Nakashima, K. Induction of apoptosis by Acanthopanax senticosus HARMS and its component, sesamin in human stomach cancer KATO III cells. Oncol. Rep. 7: 1213-1216 (2000)
  15. Schmolz, M.W., Sacher, F. and Aicher, B. The synthesis of Rantes, G-CSF, IL-4, IL-5, IL-6, IL-12 and IL-13 in human whole-blood cultures is modulated by an extract from Eleutherococcus senticosus L. roots. Phytother. Res. 15: 268-270 (2001) https://doi.org/10.1002/ptr.746
  16. Steinmann, G.G., Esperester, A. and Joller, P. Immunopharmacological in vitro effects of Eleutherococcus senticosus extracts. Arzneimittelforschung 51: 76-83 (2001)
  17. Cheng, X.J., Li, P.Z., Sheng, X.H., Li, B.J. and Zhu, C.L. Antitumor and immunological activities of Acanthopanax senticosus (Rupr. et Maxim.) Harms polysaccharides. Chinese J. Cancer 3: 191-193 (1984)
  18. Yoon, T.J., Yoo, Y.C., Lee, S.W., Shin, K.S., Choi, W.H., Hwang, S.H., Ha, E.S., Kim, S.H., Joe, S.K. and Park, W.M. Antitumor activity of Acanthopanax senticosus extract and its possible immunological mechanism of action. J. Ethnopharmcol. In press (2003)
  19. Ha, E.S., Hwang, S.H., Yu, K.W., Shin, K.S., Cho, H.M., Kim, C.H., Park, W.M. and Yoon, T.J. Immunostimulation activity of the crude polysaccharides fractionated from Eleutherococcus senticosus and its application to prevent of tumors by combination therapy with cisplatin. Yakhak Hoeji 47: 159-166 (2003)
  20. Ratcliffe, W. A., Challand, G.S. and Ratcliffe, J.G. A critical evaluation of separation methods in radiommunoassay for total triiodothyronine and thyroxine in unextracted human serum. Ann. Clin. Biochem. II: 224-229 (1974)
  21. Granstrom, M., Wretlind, B., Markman, B., Pavlovskis, O.R. and Vasil, M.L. Enzyme-linked immunosorbent assay for detection of antibodies to Pseudomonas aeruginosa exoproteins. Eur. J. Clin, Microbiol. 4: 197-200 (1985) https://doi.org/10.1007/BF02013597
  22. Kim, J.B., Barnard, G.J., Collins, W.P., Kohen, E, Lindner, H.R and Eshhar, Z. Measurement of plasma estradiol-17 beta by solidphase chemiluminescence immunoassay. Clin. Chem. 28: 1120-1124 (1982)
  23. Diamandis, E.P., Bhayana, v., Conway, K. and Reichstein, E. Papanastasiou-Diamandis, A. Time-resolved fluoroimmunoassay of cortisol in serum with a europium chelate as label. Clin. Biochem. 21: 291-296 (1988) https://doi.org/10.1016/S0009-9120(88)80083-3
  24. Izquierdo, J.M., Sotorrio, P. and Quiros, A. Enzyme immunoassay of thyroxin with a centrifugal analyzer. Clin, Chem. 28: 123-125 (1982)
  25. Park, M.L, Kim, YK., Kim, C.K. and Kim, J.B. Development of competitive enzyme-linked immunosorbent assay for the measurenment of total triiodothyronine in Serum. J. Korean Soc. Endocrinol. II: 447-454 (1996)
  26. Yoon, T.J., Yoo, Y.C., Kang, T.B., Kim, S.H., Kim, K.S. and Kim, J.B. Production of monoclonal antibodies specific to Korean Mistletoe lectin (KML-C) and their characterization. Yakhak Hoeji 45: 180-189 (2001)
  27. Schantz, S.P., Brown, B.W., Lira, E., Taylor, S.L. and Beddingfield, N. Evidence for the role of natural immunity in the control of metastatic spread of head and neck cancer. Cancer Immunol. Immunother. 25: 141-148 (1987)