Conformational ensemble-guided hapten design for improved immunorecognition of phenylbutazone in herbal tea by...

Phenylbutazone is a non-steroidal anti-inflammatory drug (NSAID) prohibited in food-producing animals due to its potential risks to food safety, requiring rapid and reliable detection methods. In this study, four haptens derived from phenylbutazone were rationally designed and evaluated for antibody production and lateral flow immunoassay (LFIA) development. In addition to conventional static similarity analysis, a conformational ensemble-based approach was introduced to better capture molecular flexibility. Although the haptens exhibited comparable structural similarity and physicochemical properties, significant differences in immunoreactivity were observed. Further recognition-region analysis suggested that linker design mainly modulated whole-molecule conformational diversity and epitope exposure without markedly disrupting the conserved phenylbutazone-like scaffold. Hapten C showed the best overall immunoreactivity and was therefore selected for antibody production and gold nanoparticle-based LFIA (GNPs-LFIA) development, achieving a cut-off value of 100 ng/mL, a limit of detection (LOD) of 4.29 ng/mL, and a working range of 5-80 ng/mL. The assay showed high specificity, satisfactory accuracy, and good agreement with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in real sample analysis. This study provides new insights into hapten design and offers a rapid method for phenylbutazone detection in complex matrices.