The detection of phosphorylated tau (p-tau) levels in clinical samples is of extreme importance for the detection of Alzheimer’s Disease (AD) as well as other neurodegenerative diseases. Recent reports show that detecting low levels of p-tau in plasma can be used as a reliable biomarker for detecting AD prior to the onset of memory loss. The ability to detect such low levels of p-tau is dependent on antibodies specific to the post translationally modified protein. However, the need for reliable phospho-site specific antibodies persists due to a lack of approaches for identifying monoclonal antibodies and characterizing non-specific binding. Here, we report a novel approach using the principles of yeast biopanning to create a robust platform that uses synthetic peptides as target antigens. Using peptides as antigens enables screening antibodies against defined post-translational modification sites, particularly for targeting intrinsically disordered proteins such as the human tau protein. To readily assess yeast binding and distinguish non-specific binding, we developed bi-directional expression vectors that allow antibody fragment surface display and intracellular fluorescent protein expression. We show that our platform can specifically and robustly detect a specific site within the p-tau target peptide when compared against non-phosphorylated controls. By improving biopanning parameters, we enabled phospho-specific capture of yeast cells displaying single-chain variable region fragments (scFvs) against p-tau with a wide range of affinities (K_D = 0.2 to 60 nM). These results demonstrate that yeast biopanning can robustly capture yeast cells based on phospho-site specific antibody binding, opening doors for facile identification of high-quality monoclonal antibodies.

中文翻译：

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用于检测与 tau 中位点特异性磷酸化的抗体结合的酵母生物淘选

临床样本中磷酸化 tau (p-tau) 水平的检测对于阿尔茨海默病 (AD) 以及其他神经退行性疾病的检测极为重要。最近的报告表明，检测血浆中低水平的 p-tau 可用作在记忆丧失开始之前检测 AD 的可靠生物标志物。检测如此低水平的 p-tau 的能力取决于翻译后修饰蛋白的特异性抗体。然而，由于缺乏识别单克隆抗体和表征非特异性结合的方法，对可靠的磷酸化位点特异性抗体的需求仍然存在。在这里，我们报告了一种使用酵母生物淘选原理的新方法，以创建一个使用合成肽作为靶抗原的强大平台。使用肽作为抗原可以筛选针对特定翻译后修饰位点的抗体，特别是针对诸如人类 tau 蛋白等固有无序蛋白质的抗体。为了轻松评估酵母结合和区分非特异性结合，我们开发了双向表达载体，允许抗体片段表面展示和细胞内荧光蛋白表达。我们表明，与非磷酸化对照相比，我们的平台可以特异性且稳健地检测 p-tau 靶肽内的特定位点。通过改进生物淘选参数，我们能够对具有广泛亲和力（K 特别是用于靶向内在无序的蛋白质，例如人类 tau 蛋白。为了轻松评估酵母结合和区分非特异性结合，我们开发了双向表达载体，允许抗体片段表面展示和细胞内荧光蛋白表达。我们表明，与非磷酸化对照相比，我们的平台可以特异性且稳健地检测 p-tau 靶肽内的特定位点。通过改进生物淘选参数，我们能够对具有广泛亲和力（K 特别是用于靶向内在无序的蛋白质，例如人类 tau 蛋白。为了轻松评估酵母结合和区分非特异性结合，我们开发了双向表达载体，允许抗体片段表面展示和细胞内荧光蛋白表达。我们表明，与非磷酸化对照相比，我们的平台可以特异性且稳健地检测 p-tau 靶肽内的特定位点。通过改进生物淘选参数，我们能够对具有广泛亲和力（K 我们开发了允许抗体片段表面展示和细胞内荧光蛋白表达的双向表达载体。我们表明，与非磷酸化对照相比，我们的平台可以特异性且稳健地检测 p-tau 靶肽内的特定位点。通过改进生物淘选参数，我们能够对具有广泛亲和力（K 我们开发了允许抗体片段表面展示和细胞内荧光蛋白表达的双向表达载体。我们表明，与非磷酸化对照相比，我们的平台可以特异性且稳健地检测 p-tau 靶肽内的特定位点。通过改进生物淘选参数，我们能够对具有广泛亲和力（K_D = 0.2 至 60 nM)。这些结果表明，酵母生物淘选可以基于磷酸位点特异性抗体结合有效地捕获酵母细胞，从而为轻松识别高质量的单克隆抗体打开大门。