Tildipirosin-Based Broad-Spectrum Antibody Preparation to Overcome Steric Hindrance and Application in a Macrolide Immunochromatographic Assay

Abstract In the livestock industry, the concurrent use of tylosin (TYL), tildipirosin (TIP), and tilmicosin (TIL) necessitates broad-spectrum detection. We propose a design strategy for immunogens and coated antigens to overcome issues of steric hindrance and affinity imbalance. Using TIP, which possesses a nitrogen-containing piperidine side chain, as a template for computer-aided hapten design, we successfully engineered a monoclonal antibody (mAb-2F12) with a spacious binding pocket. Molecular docking results show that the macrolide skeleton shared by the three analytes is stabilized by hydrogen bonds with H ASP:116 and H TYR:48, while TIP recognition additionally relies on extensive hydrophobic interactions with the binding pocket, allowing the pocket to accommodate the large side chain of TIP. To eliminate antibody recognition bias, the heterologous coating of the antigen was used to modulate the competitive equilibrium, thereby achieving broad-spectrum recognition of TIP, TYL, and TIL (IC50: 0.06–0.09 ng/mL). A gold nanoparticle immunochromatographic assay (GICA) was developed for water, soil, milk, and honey (visual detection limit: 0.1–5 ng/mL), with results validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This work demonstrates that by combining rational design of haptens and coat antigens, it is possible to achieve high-affinity, broad-spectrum recognition of contaminants within a single antibody-based immunoassay system, providing a new strategy for multiplex residue sensing.