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.

中文翻译：

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用于生物样品中甲苯噻嗪快速筛查的合理半抗原设计、抗体制备和免疫测定开发


甲苯噻嗪 （XYL） 是一种阿片类药物中的非法掺假物，是一种经批准的兽用镇静药物，已被滥用、误用和残留在食品样品中，危害人们的健康，导致药物助长犯罪甚至死亡。以抗体为核心生物材料的免疫分析可实现对 XYL 的高灵敏度和快速检测原位筛选目的。在这里，我们合理设计了四个具有不同间隔臂的新型 XYL 半抗原，以产生具有高亲和力和特异性的抗体。获得 10 个单克隆抗体 （mAb），mAb 7H5 与 IC₅₀ 的高亲和力为 0.23 ng ^mL-1，而对其他 8 个类似物的交叉反应性可忽略不计。建立了一种基于异源半抗原的高灵敏度间接竞争性 ELISA （icELISA） 和侧流免疫测定 （LFIA），用于检测一系列人尿液和食物样品中的 XYL。icELISA 的检测限 （LOD） 为 0.10-2.24 μg ^L-1，在不同样品中的回收率为 83.5% 至 128.6%，CV 低于 15.8%，而 LFIA 的视觉检测限 （vLOD） 为 0.10-1.80 μg ^L-1，临界值为 0.60-4.80 μg ^L-1。此外，还探讨了 mAb 的分子识别机制。该研究首次为快速筛查人尿液和食物样品中的 XYL 提供了强大的工具。