Development ofHigh-Affinity Monoclonal Antibodiesfor Aflatoxin M1 Detection in Milk

Aflatoxin M1 (AFM1) in milk poses significant health risks due to its carcinogenicity and resistance to thermal processing. Here, we applied a computer-aided hapten design strategy integrating density functional theory, molecular docking, and molecular dynamics simulations to engineer four AFM1 haptens with tailored spacer arms (C2, C4, C6, and phenyl). A high-affinity monoclonal antibody (10C1, IC50 = 0.015 ng/mL) was generated. Based on this antibody, an icELISA (LOD = 0.0017 ng/mL, meeting EU standards of 0.05 ng/mL) and a gold nanoparticle-based LFIA (LOD = 0.3 ng/mL, meeting China/US/Russia standards of 0.5 ng/mL) were developed for laboratory and on-site detection, respectively. This strategy facilitates rational hapten optimization and efficient generation of high-affinity antibodies for AFM1 monitoring.