Development of Highly Sensitive Immunochromatography Using Time-Resolved Fluorescence Microspheres for Amantadine Detection

Amantadine (AMA), commonly used to treat viral infections in livestock and poultry, has been banned owing to its potential hazards to human well-being. To detect unauthorized AMA usage in livestock, we developed a polyclonal antibody with a high affinity for the specific recognition of AMA through a rational design based on a structure similar to AMA and revealed the availability of the hapten design by computational chemistry analysis. Using this antibody, we established a highly responsive time-resolved fluorescence immunochromatographic assay (TRFICA). The visual detection limit of the assay is 0.6 μg/kg, and the quantitative detection limit is 0.05 μg/kg. The TRFICA also showed good recovery rates ranging from 94.5 to 109.9%, with variability coefficients not exceeding 10%. The outcomes of undisclosed sample examinations aligned with those of HPLC-MS/MS analyses, indicating that this approach can function as an ideal screening and monitoring tool for detecting illegal AMA in chicken muscle.