Analytical and Bioanalytical Chemistry

, Volume 385, Issue 6, pp 1068–1074 | Cite as

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

Original Paper

Abstract

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL−1 with a limit of detection (LOD) of 2 ng mL−1 (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL−1 and the LOD was1 ng mL−1.

Keywords

Immunoassay/ELISA Bioanalytical methods Electroanalytical methods Toxin–alkaline phosphatase conjugate Seafood toxin 

References

  1. 1.
    Kreuzer MP, O’Sullivan CK, Guilbault G (1999) Anal Chem 71:4198–4202CrossRefGoogle Scholar
  2. 2.
    Yang CC, Han KC, Lin TJ, Tsai WJ, Deng JF (1995) Hum Exp Toxicol 14:446–450CrossRefGoogle Scholar
  3. 3.
    Nunez-Vasquez EJ, Yotsu-Yamashita M, Sierra-Beltran AP, Yasumoto T (2000) Toxicon 38(5):729–734CrossRefGoogle Scholar
  4. 4.
    Lin SJ, Hwang DF (2001) Toxicon 39(4):573–579CrossRefGoogle Scholar
  5. 5.
    How CK, Chern CH, Huang YC, Wang LM (2003) Am J Emerg Med 21(1):51–54CrossRefGoogle Scholar
  6. 6.
    Watters MR (1995) Clinical Neurol Neuros 97:119–124CrossRefGoogle Scholar
  7. 7.
    Alcarez A, Whipple RE, Gregg HR, Andresen BD, Grant PM (1999) Forsensic Sci Int 99:35–45CrossRefGoogle Scholar
  8. 8.
    Sakamomoto M, Ogata T (1996) Toxicon 34(10):1101–1105CrossRefGoogle Scholar
  9. 9.
    Rivera VR, Poli MA, Bignami GS (1995) Toxicon 33(9):1231–1237CrossRefGoogle Scholar
  10. 10.
    Yotsu M, Mebs D (2001) Toxicon 39:1261–1263CrossRefGoogle Scholar
  11. 11.
    Powell CL, Doucette GJ (1999) Nat Toxins 7:393–400CrossRefGoogle Scholar
  12. 12.
    Kawatsu K, Shibata T, Hamano Y (1999) Toxicon 37:325–333CrossRefGoogle Scholar
  13. 13.
    Kreuzer MP, Pravda M, O’Sullivan CK, Guilbault GG (2002) Toxicon 40:1267–1274CrossRefGoogle Scholar
  14. 14.
    O’Leary MA, Schneider JJ, Isbister GK (2004) Toxicon 44:549–553CrossRefGoogle Scholar
  15. 15.
    Shoji Y, Yamashita MY, Miyazawa T, Yasumoto T (2001) Anal Biochem 290:10–17CrossRefGoogle Scholar
  16. 16.
    Kawatsu K, Hamano Y, Yoda T, Terano Y, Shibata T (1997) Jap J Med Sci Biol 50:133–150Google Scholar
  17. 17.
    Noguchi T, Mahmud Y (2001) J Toxicol-Toxin Rev 20:35–50CrossRefGoogle Scholar
  18. 18.
    Vetcha S, Wilkins E, Yates T (2002) Biosens Bioelectron 17:901–907CrossRefGoogle Scholar
  19. 19.
    Lombardi VC, Schooley DA (2004) Anal Biochem 331(1):40–45Google Scholar
  20. 20.
    Cagnini A, Palchetti I, Lionti I, Mascini M, Turner APF (1995) Sens Actuators B 24(1–3):85–89CrossRefGoogle Scholar
  21. 21.
    Hernandez S, Palchetti I, Mascini M (2000) Int J Environ Anal Chem 78:263–278Google Scholar
  22. 22.
    Yamashita MY, Sugimoto A, Takai A, Yasumoto T (1999) J Pharmacol Exp Therap 289(3):1688–1696Google Scholar
  23. 23.
    Alarcon SH, Micheli L, Palleschi G, Compagnone D (2004) Anal Lett 37(8):1545–1559CrossRefGoogle Scholar
  24. 24.
    Warwick MJ (1996) Standardisation of immunoassay. In: Brian L (eds) Immunoassay, a practical guide. Taylor & Francis, London, UK, p 160Google Scholar
  25. 25.
    Giraudi G, Rosso I, Baggiani C, Giovandoli C, Vanni A, Grassi G (1999) Anal Chim Acta 392(1):85–94CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Dipartimento di Scienze e Tecnologie ChimicheUniversità di Roma Tor VergataRomeItaly

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