Two binding epitopes modulating specificity of immunoassay for β-agonist detection: Quantitative structure–activity relationship

A growing number of β -agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many β -agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 β -agonists and analogues. Holographic and three-dimensional quantitative structure–activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on β -agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert -butyl at C-2′ epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of β -agonist haptens.