Epitope-Directed Antibody Development and Enhancement for Broad-Spectrum Fentanyl-Class Substance Detection: From...

The abuse of fentanyl and its analogues poses a severe threat to public health, making the development of robust detection tools crucial for effective surveillance. However, the whole category control of fentanyl makes it tough to monitor and regulate, as producers exploit extensive structural modifications to evade detection. To address this, we employed an epitope-directed hapten design strategy, systematically targeting three distinct regions of the fentanyl core structure. After immunization and hybridoma technology, three monoclonal antibodies (mAbs) with nanomolar affinity and tunable cross-reactivity profiles were generated. Furthermore, homology modeling and molecular docking elucidated the structural basis of their distinct binding patterns, establishing a clear link between hapten design and the antibody recognition function. Finally, leveraging these mAbs, we developed a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the simultaneous detection of 32 fentanyl analogues (IC₅₀ value = 0.072-101.7 ng/mL). Furthermore, a rapid gold nanoparticle-based immunochromatographic assay (GICA) with a cutoff value of 5 ng/mL was established and demonstrated its utility as a robust on-site tool. Both assays showed excellent accuracy in spiked human urine and hair samples, with recovery rates of 80.20-118.65%. This work provides not only a powerful tool for the surveillance of fentanyl analogues in complex biological matrices but also a rational hapten design framework for generating antibodies with tunable properties.