A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples

Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin‐tetrodotoxin‐formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG1,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin (TTX), with an estimated affinity of 1.2 × 108 L/M. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting TTX were developed using this MAb. A direct CIEIA using alkaline phosphatase‐labeled MAb detected TTX with sensitivities at IC50 and IC20 of 6‐7 ng/ml and 2‐3 ng/ml, respectively. The accuracy of the direct CIEIA was comparable with the high‐performance liquid chromatography (HPLC) and the mouse bioassay systems, but the direct CIEIA exhibited greater sensitivity. The direct CIEIA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.