Antibody Affinity Modulation Mechanisms via Benzene Ring Spacer Arm Conjugation Sites (Ortho, Meta, Para) in Coating Hapten Design

Benzene ring-based spacer arms have great application potential in hapten design for preparing high-affinity antibodies against small molecules. However, current studies have not systematically reported how different benzene ring spacer arm positions in the hapten regulate the antibody affinity to the coating antigen. In this work, 12 unique coating haptens with ortho, meta, and para benzene ring spacer arm positions were synthesized and coupled to carrier proteins to form coating antigens. This was done using 2-nitrobenzaldehyde derivatized semicarbazide (linear molecule), 3-amino-2-oxazolidinone (cyclic molecule), 1-aminohydantoin (cyclic molecule), and 3-amino-5-morpholinomethyl-2-oxazolidinone (bicyclic molecule) as targets to determine the influence of the spacer position at the benzene ring on the antibody affinity for the coating antigen. First, we evaluated the performance of eight prepared monoclonal antibodies (mAbs) by an indirect enzyme-linked immunosorbent assay (iELISA), indirect competitive ELISA (icELISA), and lateral flow immunoassay (LFA). Moreover, the interaction forces between antibodies and different coating haptens were examined by molecular docking to investigate the aforementioned regulatory mechanisms. Specifically, we confirmed that in coating hapten design (linear/cyclic molecule), the position (meta, para) of benzene ring spacer arms used for carrier protein conjugation enhanced antigen-antibody recognition via π-π or π-alkyl interactions, thereby modulating antibody affinity toward the derivatized compounds. Although the benzene ring spacer arm minimally contributed to antigen-antibody recognition in complex small molecules (e.g., bicyclic compounds), its primary role was to maintain a critical hapten-carrier protein distance and prevent steric hindrance. Finally, highly sensitive LFAs for detecting nitrofuran residues in fish and shrimp were developed, providing a versatile platform that can be extended to other chemical compounds.