Broad-spectrum antibody against thiazides and degraded salamide and immunoassay establishment for simultaneous detection

Abstract

The illegal additive thiazides and degraded salamide in food supplements poses significant health risks and regulatory challenges. However, the structural differences between thiazides and salamide complicate the hapten design for immunoassay detection. In this study, we first outlined the common skeleton of thiazides and their shared epitopes with salamide through molecular overlap and atomic charge distribution analysis. Several haptens were rationally designed based on the private epitope of salamide and the shared epitopes with thiazides, assisted by computer-aided chemical analysis. Subsequently, the resultant monoclonal antibody exhibited broad specificity for thiazides (IC₅₀ value = 21.43 to 358.76 ng/mL) and salamide (IC₅₀ value = 0.76 ng/mL). The immunoassay affinities of 5 of these thiazides are reported for the first time. Further elucidation of the intrinsic recognition mechanism was achieved through homology modeling and molecular docking, clarifying the antibody's diverse recognition spectrum. Finally, we established a rapid, reliable colloidal gold-based immunochromatography strip for simultaneous detecting total thiazides and degraded salamide in food supplements and surface water sample for the first time. The cut-off value was as low as 7.5 ng/mL, which was 32 times more sensitive than that of established immunoassays for thiazides.