Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China

Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2010 Nov;27(11):1589-97. doi: 10.1080/19440049.2010.504237.

Abstract

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX-keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10(-8) mol l(-1). The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 10(6); the reference working concentration was 1:1.2 × 10(5). By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose-response curve of TTX concentration was in range 5-500 ng ml(-1). The limit of detection was 5 ng ml(-1) according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml(-1). The recoveries from TTX spiked samples were 79.5-109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Murine-Derived / immunology
  • Antibody Affinity
  • China
  • Cross Reactions
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Food Contamination* / prevention & control
  • Food Inspection / methods*
  • Foodborne Diseases / prevention & control
  • Haptens
  • Hemocyanins
  • Limit of Detection
  • Male
  • Pacific Ocean
  • Reproducibility of Results
  • Seafood / analysis*
  • Seafood / toxicity
  • Species Specificity
  • Tetraodontiformes* / classification
  • Tetrodotoxin / analysis*
  • Tetrodotoxin / immunology

Substances

  • Antibodies, Monoclonal, Murine-Derived
  • Haptens
  • Tetrodotoxin
  • Hemocyanins
  • keyhole-limpet hemocyanin