Abstract: A microsphere-based fluorescence immunochromatographic assay was established for the detection of tetrodotoxin. A binding site protection procedure was developed for antibody labeling with fluorescent microspheres, which prevented damage to the antibody binding site and consequently enhanced the fluorescence signal. The whole process of binding and separation was efficiently completed on an affinity spin column. The sensitivity of the antibody-microsphere conjugate was nine times higher than that prepared by direct labeling. The immunochromatographic assay exhibited a linear range from 2 to 200 ng/mL for the detection of tetrodotoxin (y = ?0.15lnx + 0.95, R2 = 0.98) with an IC50 of 18.4 ng/mL. The limit of detection (LOD) for tetrodotoxin was 10 μg/kg in puffer with an average relative standard deviation (RSD) of less than 25% (n = 6). The assay method could be applied on heat-processed products without the need for large-scale equipment. The whole analysis process took only 15 min for each sample.

Key words: tetrodotoxin, binding site protection, fluorescent microspheres, immunochromatography