Development of an Ultrasensitive Monoclonal Antibody against Butralin Based on a Novel Hapten Design Strategy

Butralin (BL) is a dinitroaniline herbicide commonly applied in agriculture to control annual grasses and broadleaf weeds. However, its environmental persistence and potential toxicity to nontarget organisms, including aquatic species and humans, have raised serious concerns. Reliable monitoring of BL residues is therefore crucial for ensuring environmental and food safety. In this study, a novel hapten was synthesized by introducing an ethylenediamine group at the C1 position of 4-<i>tert</i>-butyl-2,6-dinitrobenzene, followed by spacer arm extension via succinic anhydride acylation. Using this hapten, a specific monoclonal antibody (mAb) against BL was generated, exhibiting the highest affinity reported to date. Based on this mAb, an optimized indirect competitive ELISA (ic-ELISA) was established, providing excellent sensitivity with an IC₅₀ of 0.385 ng/mL and a linear detection range (IC₂₀-IC₈₀) of 0.012-1.24 ng/mL. The mAb also demonstrated remarkable specificity for BL, with negligible cross-reactivity toward structurally related analogs, confirming its potential for developing sensitive and selective immunoassays for residue monitoring in environmental and food samples.