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.
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.
Tao
J
J
National Institute for Nutrition and Food Safety, Chinese CDC, Beijing 100021, China.
Wei
W J
WJ
Nan
L
L
Lei
L H
LH
Hui
H C
HC
Fen
G X
GX
Jun
L Y
LY
Jing
Z
Z
Rong
J
J
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology; D000915:Antibody Affinity; D002681:China; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005506:Food Contamination / Q000517:prevention & control; D005513:Food Inspection / Q000379:methods*; D005517:Foodborne Diseases / Q000517:prevention & control; D006241:Haptens; D006433:Hemocyanins; D057230:Limit of Detection; D008297:Male; D010140:Pacific Ocean; D015203:Reproducibility of Results; D017747:Seafood / Q000032:analysis* / Q000633:toxicity; D013045:Species Specificity; D023721:Tetraodontiformes / Q000145:classification; D013779:Tetrodotoxin / Q000032:analysis* / Q000276:immunology
D058846:Antibodies, Monoclonal, Murine-Derived; D006241:Haptens; D013779:Tetrodotoxin; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin