Antibody Recognition Profile-Aided Hapten Design to Modulate Antibody Generation with Anticipated Performance for Immunoassay Development

The hapten design to chemical compound currently considered only structural aspects of targets may suffer from the failure of antibody generation with anticipated performances, especially for broad-specific antibodies. To address the problem, this study proposed a novel strategy, named antibody recognition profile-aided hapten design (ARPHD), based on clues from both reported antibodies, haptens, and targets after uncovering antibody recognition profiles using fluorfenicol (FF) and fluorfenicol amine (FFA) as model analytes in this work. Specifically, we confirmed that the fluorine atom promoted the generation of antibodies to FFA, while the –COCHCl2moiety was unfavorable for inducing broad-specific antibodies to FF and FFA. Based on the structural information from ARPHD, four novel haptens without –COCHCl2and containing a fluorine atom are intentionally designed, then identified by computational chemistry and animal immunization, successfully inducing antibodies to FF and FFA with uniform IC50values of 3.09 and 3.75 ng mL–1. The explanation of molecular mechanisms from the obtained antibodies has supported the scientific base behind ARPHD, and we also found that the light chain of the antibody contributed an important role in differential recognition of the antibody. Finally, an indirect competitive ELISA (icELISA) was developed for the simultaneous detection of FF and FFA in river water and animal-derived food with a LOD of 2.24–14.6 μg kg–1, which has never been achieved before. The study demonstrated that the ARPHD we proposed could rationally guide the design of haptens that modulate the generation of antibodies with appreciated performances and easily be extended to other chemical compounds as a versatile platform.