中文摘要：霉酚酸（MPA）是一种免疫抑制剂，常用于预防移植患者的器官排斥反应。MPA监测因其治疗窗口小而备受推崇。本研究使用噬菌体展示肽库选择与MPA特异性重组抗体片段（Fab）结合的环肽，并模拟MPA的行为。经生物淘洗，分离并测试了几种噬菌体展示肽，以确认它们在基于噬菌体的竞争性免疫分析中的表位模拟性质。在确定最佳MPA模拟物（具有二硫键约束环的ACEGLYAHWC）后，测试了几种免疫分析方法，并开发了含有肽序列和生物发光酶NanoLuc的重组融合蛋白。重组融合后可直接用作竞争性免疫分析中的示踪剂，而无需二级抗体或进一步标记。生物发光传感器使用链霉亲和素偶联磁珠固定生物素化Fab抗体，检测MPA的检测限为0.26ng mL(-1)， IC50为2.9+/-0.5ng mL(-1)。该生物传感器对MPA表现出良好的选择性，并应用于分析临床样本中健康和MPA治疗患者的免疫抑制药物，然后通过液相色谱结合二极管阵列检测进行验证。
外文摘要：Mycophenolic acid (MPA) is an immunosuppressant drug commonly used to prevent organ rejection in transplanted patients. MPA monitoring is of great interest due to its small therapeutic window. In this work, a phage-displayed peptide library was used to select cyclic peptides that bind to the MPA-specific recombinant antibody fragment (Fab) and mimic the behavior of MPA. After biopanning, several phage-displayed peptides were isolated and tested to confirm their epitope-mimicking nature in phage-based competitive immunoassays. After identifying the best MPA mimetic (ACEGLYAHWC with a disulfide constrained loop), several immunoassay approaches were tested, and a recombinant fusion protein containing the peptide sequence with a bioluminescent enzyme, NanoLuc, was developed. The recombinant fusion enabled its direct use as the tracer in competitive immunoassays without the need for secondary antibodies or further labeling. A bioluminescent sensor, using streptavidin-coupled magnetic beads for the immobilization of the biotinylated Fab antibody, enabled the detection of MPA with a detection limit of 0.26 ng mL(-1) and an IC50 of 2.9 +/- 0.5 ng mL(-1). The biosensor showed good selectivity toward MPA and was applied to the analysis of the immunosuppressive drug in clinical samples, of both healthy and MPA-treated patients, followed by validation by liquid chromatography coupled to diode array detection.
外文关键词：PHAGE DISPLAY；IMMUNOASSAY；ASSAY；SELECTION；MOFETIL；EMIT
作者：Luque-Uria, A；Peltomaa, R；Nevanen, TK；Arola, HO；Iljin, K；Benito-Pena, E；Moreno-Bondi, MC
作者单位：Univ Complutense Madrid；Univ Turku；VTT Tech Res Ctr Finland Ltd
期刊名称：ANALYTICAL CHEMISTRY
期刊影响因子：6.785
出版年份：2021
出版刊次：29
点击下载：重组肽模拟NanoLuc示踪剂用于霉酚酸的灵敏免疫检测