A highly specific antibody profiled by hapten prediction and its application in an immunoassay for Anilazine

Anilazine (Ani) is an endobronchial fungicide with a wide bactericidal spectrum and Ani contamination in the environment and food has become an important safety problem. In this study, we established a rapid detection method for Ani with high specificity, and high sensitivity and important applications in the field. We evaluated three haptens of Ani and their Electrostatic Potential (ESP) surfaces whereby the three haptens were first analyzed by computer-aided technology combined with Visual Molecular Dynamics (VMD) and Multiwfn 3.7 (dev) code. The molecular structure overlapped at the compound's lowest energy conformation was carried out using the Discovery Studio molecular superposition dialog box. And a highly-specific antibody (2B3) was obtained by matrix effect enhancement screening with an IC₅₀ value of 0.52 ng/mL. A colloidal gold immunochromatography assay (CIA) was first established for the detection of Ani in tomato, wheat, water, and soil samples and the vLOD (Visual limits of detection) values were 10, 20, 5, 10 ng/mL, and the elimination values were 50, 200, 20, 100 ng/mL, respectively. These values met the maximum residue limit requirements for Ani found in the Chinese GB 2763-2021. The CIA strip recovery test was 91.5 % - 99.7 %, the coefficient of variation (CV) was 1.45 % - 6.02 %, and the accuracy was verified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. The CIA stability test confirmed that the strips could be stored stably for some time under specific conditions. Therefore, the rapid detection method established in this study was accurate and reliable, as well as having the potential for key screening and monitoring of Ani in relevant samples.