Rapid and highly sensitive enzyme immunoassay for quantitative determination of tetrodotoxin

A monoclonal antibody against tetrodotoxin (TTX) was obtained from Balb/c mice immunized with TTX-bovine serum albumin (BSA) conjugate. The monoclonal antibody was highly specific for TTX and had no cross-reaction to tetrodonic acid, which is a TTX derivative, or gonyautoxins, although a minor cross-reaction to anhydro-tetrodotoxin was observed. The monoclonal antibody neutralized the lethal activity of TTX. By using the monoclonal antibody, a rapid and highly sensitive competitive enzyme immunoassay (EIA) for quantitative analysis of TTX was developed. By the competitive EIA system, TTX can be determined quantitatively in about 30 min (90 min are required if the time for preparation of the solid-phase antigen was included), and the working range for quantitative analysis of TTX was 2-100 ng/ml. In recovery tests and examinations of TTX samples, results of the mouse bioassay and EIA analyses correlated well (r = 0.987). Moreover, it was demonstrated that low concentrations of TTX, which could not be detected by the mouse bioassay, could be determined quantitatively by the competitive EIA.