Establishment of a new ELISA system for measuring the concentration of the puffer fish toxin tetrodotoxin

Abstract

Even in these days, food poisoning due to tetrodotoxin (TTX) still occurs, but there are no clinical trials of methods to measure TTX concentration. In this study, we attempted to establish a new enzyme-linked immunosorbent assay (ELISA) system for assessing TTX concentration. First, we established six patterns of noncompetitive ELISA to generate calibration curves, and we determined the best two antibodies for measuring TTX concentration. Next, the novel assay system was used for fourteen simultaneous measurements of equivalent TTX concentrations (100 μg/mL) within different matrices (citrate buffer, urine, or serum). Using this ELISA system, we determined the concentration of TTX diluted in citrate buffer with high accuracy and precision. However, the TTX concentration in urine was low (37.36 μg/mL), whereas that in serum was high (249.86 μg/mL). To correct the effect of interfering components in each biological sample, we regenerated calibration curves using serial dilutions of the TTX standard prepared using urine or serum. Then, the TTX concentration in urine was increased (111.86 μg/mL), but that in serum remained high (162.92 μg/mL). Finally, deproteinization treatment increased the TTX concentration in serum (111.29 μg/mL) measured by this new ELISA system. Although this ELISA system still needs further improvement, for measuring the TTX concentration in biological samples, this method would be useful for laboratory medicine to some extent.