Shang Jing, Zrazhevskiy Pavel, Postupna Nadia, Keene C Dirk, Montine Thomas J, Gao Xiaohu
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
Sci Rep. 2015 Sep 2;5:13651. doi: 10.1038/srep13651.
In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the "Alzheimer's disease" cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics.
细胞内免疫测定已成为蛋白质表达分析的一种有价值的工具，可作为对现有测定形式的补充。然而，由于报告酶的单重性质以及替代测定形式的技术复杂性，对单个样本进行全面的分子表征已被证明具有挑战性且不切实际。在此，我们描述了一种简单且稳健的方法，用于在同一完整样本上进行多重蛋白质表达谱分析，采用一种特性明确的碱性磷酸酶来准确量化所有感兴趣的靶标，同时通过实施DNA编程释放机制来分离靶标结合报告分子的子集，从而克服基于酶的技术的基本局限性。从本质上讲，这种方法将同一样本的多靶标标记转化为一组以并行自洽方式进行的孤立单重测量。作为原理验证，在培养的HeLa细胞上展示了对三种模型蛋白的多重检测，并在福尔马林固定石蜡包埋的脑组织切片中对痴呆症的两个临床相关标志物β-淀粉样蛋白和PHF- tau进行了分析，发现“阿尔茨海默病”队列中这两种标志物的丰度都有相关增加。多重细胞内免疫测定具有强大的分析能力，但技术上简单且稳健，有望实现有洞察力的同一样本蛋白质谱研究，并在生物医学研究和临床诊断中得到广泛应用。
