Extending the reach of immunoassays to optically dense specimens by using two-photon excited fluorescence polarization
Extending the reach of immunoassays to optically dense specimens by using two-photon excited fluorescence polarization
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DOI：
10.1021/ac0004761
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发表时间：
2000-11-15
影响因子：
7.4
通讯作者：
Bright, FV
中科院分区：
文献类型：
--
作者：
Baker, GA;Pandey, S;Bright, FV
荧光各向异性/偏振测量是量化生物分子/配体络合的强大工具。这些类型的测量也是各种商业均质荧光免疫测定的核心。在本文中，我们展示了双光子激发荧光各向异性 (2-PEFA) 测量作为在存在强吸收性非荧光染料的情况下量化半抗原/抗体关联的工具的强大功能。这些实验的结果表明，与传统的单光子激发荧光各向异性 (1-PEFA) 策略相比，2-PEFA 测量本质上更灵敏，并且基于 2-PEFA 的测量允许在 1-PEFA 测量完全失败的条件下直接在强吸收样品中进行准确的半抗原/抗体结合测量。总体而言，与传统的 1-PEFA 方法相比，2-PEFA 方法具有显着的优势，尤其是在强吸收样品中。 2-PEFA 还为以最少的样品制备进行更快速、更可靠的基于偏振/各向异性的测量打开了大门。
Fluorescence anisotropy/polarization measurements represent a powerful tool for quantifying biomolecule/ligand complexation. These types of measurements are also at the heart of a wide variety of commercial homogeneous fluoroimmunoassays. In this note, we demonstrate the power of two-photon excited fluorescence anisotropy (2-PEFA) measurements as a tool for quantifying hapten/antibody association in the presence of a strongly absorbing, nonfluorescent dye. The results of these experiments show that 2-PEFA measurements are intrinsically more sensitive when compared to traditional one-photon excited fluorescence anisotropy (1-PEFA) strategies and 2-PEFA-based measurements allow one to perform accurate hapten/antibody binding measurements in strongly absorbing samples directly under conditions where 1-PEFA measurements fail completely. Overall, the 2-PEFA approach offers significant advantages when compared to traditional 1-PEFA methods especially in strongly absorbing samples. 2-PEFA also opens the door to perform more rapid and reliable polarization/anisotropy-based measurements with minimal sample preparation.