用于免疫抑制剂类治疗药物监测的可视化免疫芯片构建及应用

Immunosuppressive drugs have been used for organ transplantation by inhibiting the activity of immunesystem, including cyclosporin A, tarcrolimus and sirolimus. Due to narrow therapeutic range, variable pharmacokinetics, and lack of a reliable correlation between dose and drug exposure, the application of these drugs requires regular immunosuppressant therapeutic drug monitoring (TDM) in order to arrive at the optimal dose for therapeutic efficacy as well as minimizing toxicity. In this program, a label-free colorimetric biosensor used in therapeutic drug monitoring of immunosuppressive agents are developed. Haptens are obtained by modifing the molecules of drugs using different couping agents, and immunogens are synthesized by coupling hapten with carrier proteins. Diacetylence molecules were then used to couple with hapten to act as probes and polymized to form polydiacetylene (PDA) vesicles. By immoblizationg of PDA vesicles onto an amino-modified glass slide, a colorimetric microarray are established. With the antibodies of immunosuppressive drugs as the ligand, the PDA vesicles would change from blue to red. By using avidin-HRP as signal amplification, a competitive immunoassay bisensor would be developed and used to detect immunosuppressive drugs in plasma in a rapid, high-throughput way.

免疫抑制剂药物在临床上治疗器官和骨髓移植后排斥反应的治疗窗很窄，血浆浓度过高会出现严重的肝肾毒性、高血糖、神经损害，浓度偏低时以发生排斥反应、感染、甚至死亡。该类药物的药代动力学个体差异较大，且毒性和服药量之间相关性不明确，临床上需密切监控以便及时调整治疗方案。现有的免疫检测试剂特异性差，无法区分原型药和代谢产物，且被国外生物试剂公司垄断。本项目拟通过修饰半抗原分子结构，自主研制高特异性的灵敏抗体；同时应用具有生物分子识别性能的联乙炔与半抗原偶联制得的聚联乙炔囊泡探针作为敏感元件，识别抗体后囊泡由蓝变红，同时产生荧光；样品中的待测药物将与有限的抗体竞争结合，导致显色反应/荧光强度变弱，根据变色强度与待测药物浓度呈负相关的趋势构建可视化免疫微阵列芯片，结合亲和素标记的辣根过氧化物酶放大显色响应，以快速、便捷、高通量监测血浆中的药物浓度，实现临床实时监控。

本课题首先针对根据任务书指向的三种常用免疫抑制剂药物(环孢素A、他克莫司和西罗莫司)的结构特点进行半抗原分子设计，改造和修饰，制备完全免疫原并进行了结构表征。以新西兰大白兔为免疫动物、分别制备了多克隆抗体，结果表明所得抗体产品特异性较强，灵敏度较好。项目的实施获得了自主知识产权的抗体产品，开发出了目前国内医疗市场急需的血药浓度免疫检测关键试剂，突破小分子抗体免疫原性低，制备过程复杂，抗体亲和性差，交叉反应严重等技术难题；接着将具有生物分子识别性能的联乙炔与半抗原（环孢素A）偶联制得的聚联乙炔（PDA）囊泡探针作为敏感元件，识别环孢素A抗体后囊泡由蓝变红；样品中的待测药物将与有限的抗体竞争结合，导致显色反应/荧光强度变弱, 根据变色强度与待测药物浓度呈负相关的趋势首次构建了竞争性可视化免疫传感元件。探索了该可视化传感元件的可视化和荧光检测灵敏度，以及该检测体系与半抗原分子的大小和水溶性的关联，拓展了聚联乙炔囊泡在小分子检测中的应用；最后应用所得抗体产品，利用抗原抗体反应与化学发光免疫技术和电化学免疫技术结合起来，开发适用于不同检测情况的快速分析方法和优化产品，突破了目前使用较多的仪器分析法的局限，并实际应用于大鼠口服后的血药浓度监测及药代动力学研究中。