Аннотация:Immunoassay methods are important to monitor β-agonists 25 illegally used for reducing animal fat deposition in livestock. However, there is no a simultaneous screening surveillance immunoassay for possibly occurred various β-agonist chemicals in food. In this study, using an R-(–)-salbutamol derivative as the immunizing hapten, an antibody recognizing 31 β-agonists and analogues was generated for the first time. Three-dimensional quantitative structure-activity relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the hapten spacer near C-2, as well as a chirality at C-1´, dominantly modulated the class specificity of the raised antibody. However, a hapten spacer linked at C-2´ or C-1 would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised antibody specificity spectrum. A class specificity competitive indirect enzyme linked immunosorbent assay (ciELISA) was established with an ideal recovery ranged from 81.8% to 118.3% based on the obtained antibody. With a good agreement to HPLC-MS method, the proposed ciELISA was confirmed to be reliable for the rapid surveillance screening assay of β-agonists in urine sample. This investigation will contribute to the rational design and control of the immunoassay specificity