Sensitive Rapid Fluorescence Polarization Immunoassay for Free Mycophenolic Acid Determination in Human Serum and Plasma
Sensitive Rapid Fluorescence Polarization Immunoassay for Free Mycophenolic Acid Determination in Human Serum and Plasma
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DOI：
10.1021/acs.analchem.8b00780
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发表时间：
2018-04-17
影响因子：
7.4
通讯作者：
Moreno-Bondi, Maria C.
中科院分区：
文献类型：
--
作者：
Glahn-Martinez, Bettina;Benito-Pena, Elena;Moreno-Bondi, Maria C.
本文介绍了一种新的近红外荧光染料标记的荧光偏振免疫分析法（FPIA）。建立了快速分析移植患者血浆中游离霉酚酸（MPA）的荧光偏振免疫分析方法。该方法是基于目标免疫抑制剂和一种新的发射近红外荧光染料标记的MPA和MPA-AO的抗MPA抗体的结合位点之间的荧光竞争测定。MPA的荧光类似物在629 nm（λ（max）（exc））激发时在654 nm处显示出发射，并且在磷酸盐缓冲介质中显示出良好的光化学稳定性和显著的发射量子产率（0.16）。在分析前，使用超滤从血液或血浆样品中分离游离麦考酚酸。在测量之前，将样品与5 μ g/mL抗MPA抗体和1 nM MPA-AO孵育20分钟。所开发的FPIA在PBST缓冲液中显示出0.8 ng/mL（10%结合抑制）的检测限和1.7-39 ng/mL（20%-80%结合抑制）的动态范围，符合治疗要求。通过测量与MPA联合给药的其他免疫抑制药物（环孢素A和他克莫司）以及代谢产物MPA葡萄糖醛酸苷的交叉反应性，评价免疫测定选择性。该方法已成功地应用于分析游离MPA在心脏移植患者的血液口服后，霉酚酸酯（MMF）和他克莫司，并与快速分辨率液相色谱二极管阵列检测器（RRLC-DAD）所获得的结果进行了比较。
In this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near-infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (lambda(max)(exc)) and shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 mu g/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7-39 ng/mL (20%-80% binding inhibition) in a PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart-transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus, and the results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).