Rational hapten design, antibody preparation, and immunoassay development for rapid screening xylazine in biological samples

Xylazine (XYL) is an illicit adulterant in opioids and an approved veterinary sedative drug, which has been abused, misused, and residued in food samples, endangering people health, causing drug-facilitated crimes and even death. Immunoassay used antibody as core biomaterial could to achieve highly sensitive and rapid detection screening purpose for XYL in situ. Here, we rationally designed four novel XYL haptens with different spacer arms to produce antibodies with high affinity and specificity. Ten monoclonal antibodies (mAbs) were obtained and mAb 7H5 showed a high affinity with IC₅₀ of 0.23 ng mL⁻¹ and ignorable cross-reactivity for the other eight analogs. One highly sensitive indirect competitive ELISA (icELISA) and lateral flow immunoassay (LFIA) were established based on heterologous haptens for XYL detection in a series of human urine and food samples. The limit of detections (LODs) of the icELISA were 0.10–2.24 μg L⁻¹ with recovery of 83.5 % to 128.6 % and CV below 15.8 % in different samples, while, the visual limits of detection (vLOD) of LFIA were 0.10–1.80 μg L⁻¹ with the cut-off value of 0.60–4.80 μg L⁻¹. In addition, the molecular recognition mechanism of mAbs was explored. The study provides powerful tools for rapid screening of XYL in human urine and food samples for the first time.