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Sensitive Rapid Fluorescence Polarization Immunoassay for Free Mycophenolic Acid Determination in Human Serum and Plasma
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-03-26 00:00:00 , DOI: 10.1021/acs.analchem.8b00780 Bettina Glahn-Martínez 1 , Elena Benito-Peña 1 , Francesca Salis 2 , Ana B. Descalzo 2 , Guillermo Orellana 2 , María C. Moreno-Bondi 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-03-26 00:00:00 , DOI: 10.1021/acs.analchem.8b00780 Bettina Glahn-Martínez 1 , Elena Benito-Peña 1 , Francesca Salis 2 , Ana B. Descalzo 2 , Guillermo Orellana 2 , María C. Moreno-Bondi 1
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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 (λexcmax) 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 μg/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).
中文翻译：
灵敏的快速荧光偏振免疫分析法测定人血清和血浆中的游离麦考酚酸
在本文中，我们描述了一种使用新标记-近红外荧光染料的荧光偏振免疫测定（FPIA）。已开发的FPIA方法经过优化，可快速分析移植患者血浆中的游离麦考酚酸（MPA）。该方法基于靶免疫抑制剂与新型发光近红外荧光染料标记的MPA和MPA-AO之间针对抗MPA抗体结合位点的荧光竞争测定。MPA的荧光类似物在629 nm处激发后在654 nm处显示发射（λexc max），并在磷酸盐缓冲液中显示出良好的光化学稳定性和显着的发射量子产率（0.16）。在分析之前，使用超滤从血液或血浆样品中分离出游离的霉酚酸。在测量之前，将样品与5μg/ mL的抗MPA抗体和1 nM的MPA-AO孵育20分钟。发达的FPIA在PBST缓冲液中的检出限为0.8 ng / mL（结合抑制10％），动态范围为1.7–39 ng / mL（结合抑制20％–80％），符合治疗要求。通过测量与MPA（环孢菌素A和他克莫司）联合使用的其他免疫抑制药物以及代谢产物MPA葡萄糖醛酸的交叉反应性来评估免疫分析的选择性。
更新日期：2018-03-26
中文翻译：
灵敏的快速荧光偏振免疫分析法测定人血清和血浆中的游离麦考酚酸
在本文中，我们描述了一种使用新标记-近红外荧光染料的荧光偏振免疫测定（FPIA）。已开发的FPIA方法经过优化，可快速分析移植患者血浆中的游离麦考酚酸（MPA）。该方法基于靶免疫抑制剂与新型发光近红外荧光染料标记的MPA和MPA-AO之间针对抗MPA抗体结合位点的荧光竞争测定。MPA的荧光类似物在629 nm处激发后在654 nm处显示发射（λexc max），并在磷酸盐缓冲液中显示出良好的光化学稳定性和显着的发射量子产率（0.16）。在分析之前，使用超滤从血液或血浆样品中分离出游离的霉酚酸。在测量之前，将样品与5μg/ mL的抗MPA抗体和1 nM的MPA-AO孵育20分钟。发达的FPIA在PBST缓冲液中的检出限为0.8 ng / mL（结合抑制10％），动态范围为1.7–39 ng / mL（结合抑制20％–80％），符合治疗要求。通过测量与MPA（环孢菌素A和他克莫司）联合使用的其他免疫抑制药物以及代谢产物MPA葡萄糖醛酸的交叉反应性来评估免疫分析的选择性。
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