Development of High-Affinity Monoclonal Antibodies for Aflatoxin M1 Detection in Milk.

Abstract

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.