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首页> 外文期刊>CHROMATOGRAPHY >Fluorescence Bioanalysis of Bevacizumab Using Pre-Column and Post-Column Derivatization – Liquid Chromatography After Immunoaffinity Magnetic Purification
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Fluorescence Bioanalysis of Bevacizumab Using Pre-Column and Post-Column Derivatization – Liquid Chromatography After Immunoaffinity Magnetic Purification

机译：使用预柱和柱后衍生化 - 液相色谱法荧光生物分析 - 免疫亲和力磁性净化后

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This report presents two fluorescence labeling methods for therapeutic monoclonal antibody, bevacizumab, to increase its detection sensitivity for fluorescence detection. One method is high-temperature reversed-phase LC (HT-RPLC) following post-column fluorogenic derivatization using o -phthalaldehyde with thiol. Another method is pre-column derivatization using Zenon Alexa Fluor 488 protein-tag following size-exclusion chromatography (SEC). The calibration curves of bevacizumab were 1–50 μg/mL (post-column method) and 0.1–10 μg/mL (pre-column method). Both methods showed good correlation coefficients (r~(2) > 0.991). The LOD and the LOQ of bevacizumab were, respectively, 0.13 and 0.43 μg/mL (post-column method) and 0.03 and 0.1 μg/mL (pre-column method). The sensitivities were about 2 and 10 times higher than that of native fluorescence detection. The proposed methods were applied to bevacizumab spiked human plasma samples. The bevacizumab in plasma samples was purified selectively with immunoaffinity beads and detected as a single peak using HT-RPLC or SEC with fluorescence detection.
机译：该报告呈现了治疗性单克隆抗体，贝伐单抗的两种荧光标记方法，以增加其对荧光检测的检测灵敏度。一种方法是在柱后氟因尼衍生后使用硫醇的含柱氟因尼衍生化的高温反相LC（HT-RPLC）。在尺寸排除色谱（SEC）之后，另一种方法是使用Zenon Alexa Fluor 488蛋白标签的柱衍生化。 Bevacizumab的校准曲线为1-50μg/ ml（柱后法）和0.1-10μg/ ml（柱法）。两种方法显示出良好的相关系数（R〜（2）> 0.991）。贝伐单抗的LOD和LOQ分别为0.13和0.43μg/ ml（柱后法）和0.03和0.1μg/ mL（柱状法）。比天然荧光检测的敏感性约为2％至10倍。将所提出的方法应用于贝伐单抗尖刺人血浆样品。血浆样品中的贝伐单抗被选择性地用免疫亲和性珠粒纯化，并使用HT-RPLC或SER检测为单峰，具有荧光检测。

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