The development and optimization of ELISA for the determination of tetrodotoxin*

ObjectiveTo optimize the ELISA for the determination of tetrodotoxin.MethodsA competitive enzyme-linked immunosorbent assay (ELISA) was used. In the ELISA, 100 μl antigen (1.0 μg/ml) was coated on the microtiter plate for 60 min at 37 C or over night at 4 C. The plate was then washed 3 times with PBS-T for 3-5 s each time. The optimal incubation time for monoclonal antibody (mAb), goat anti-mice IgG peroxidase conjugate and OPD were 30 min. 20 min and 10 min at 37 C, respectively.ResultsThe detection limit is 0.05 ng in each well. The curve was linear for TTX doses between 5-5 000 ng/ml (0.25-250 ng for every assay). The linear regress equation was Y = 0.30 88X — 0.17 41 (R. = 0.99 01). The average callback for TTX of muscles and gonads were 99.74% and 100.30%, respectively. The sensitivity of optimization ELISA was 5 times than traditional method and the time of 1.8 h were saved.ConclusionThe optimized ELISA is an idealmethod for the determination of tetrodotoxin.