PO.CH02.02 · 化学
用于nCounter高重数多组学面板的多步骤抗体验证
Multi-step antibody validation for nCounter high-plex multiomics panels
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
nCounter®分析系统的拓展开发现已能够从单个FFPE组织样本中同时定量超过500种蛋白和多达800种转录本。通过引入两个高重数蛋白面板并与任意预建的nCounter® RNA面板结合使用，这一新功能为免疫肿瘤学多组学研究带来了一种新颖的非破坏性FFPE检测，具有新的深度、简便性和可靠性。nCounter®平台长期以来被公认为肿瘤学和免疫学研究（包括诊断开发）的金标准。确认其在支持转化和临床应用中的性能是该功能的重要组成部分。为确保在这一复杂环境中获得高数据完整性，我们开发了一套严格的多步骤抗体验证流程。该流程始于由我们的抗体合作伙伴AbCam进行的初步IHC筛选，以确认在已知阳性和阴性组织中的特异性。这些经单重IHC验证的抗体分别偶联至nCounter兼容的UV可裂解寡核苷酸。使用90多个细胞系和来自30多种组织类型（涵盖癌变和健康FFPE样本）的100多个组织芯，评估了高重数蛋白面板的功能性能。灵敏度和特异性以细胞系可获得的正交RNA-Seq和质谱数据集为基准进行比对。磷酸特异性抗体的特异性还在磷酸酶处理实验中进行了测试，确认处理后超过90%的信号被削减。为证明这些抗体在高重数面板中的兼容性，我们展示了当我们的蛋白面板堆叠组合或独立运行时，抗体性能保持稳健。最后，跨用户和跨批次确认了重现性。这一验证框架确保了高特异性、灵敏度和重现性，能够对复杂组织样本进行可靠的多组学分析，并加速癌症生物学中的转化见解。
查看英文原文 English abstract
Expanded development on the nCounter® Analysis System now enables simultaneous quantification of over 500 proteins and up to 800 transcripts from a single FFPE tissue sample. With the introduction of two high-plex protein panels used in combination with any prebuilt nCounter® RNA Panel this new capability brings a novel non-destructive FFPE assay with new depth, simplicity and reliability for immuno-oncology multiomics research. The nCounter® platform has long been known as the gold standard for oncology and immunology studies including diagnostic development. Confirming performance in support of translational and clinical applications is essential part of this capabilityTo ensure high data integrity in this complex environment, we developed a rigorous multi-step antibody validation pipeline. The process begins with an initial IHC screening, performed by our antibody partner AbCam, to confirm specificity in known positive and negative tissues. These single-plex IHC validated antibodies are individually conjugated to nCounter-compatible UV-cleavable oligonucleotides. Functional performance of the high-plex protein panels was assessed using more than 90 cell lines and more than 100 tissue cores from over 30 tissue types featuring cancerous and healthy FFPE samples. Sensitivity and specificity are benchmarked against available orthogonal RNA-Seq and mass spectrometry datasets for the cell lines. The specificity of phospho-specific antibodies was also tested in a phosphatase treatment assay, confirming that >90% of signal is reduced with treatment.To demonstrate the compatibility of these antibodies in a high-plex panel, we show antibody performance remains robust when our protein panels are stacked together or run independently. Finally, reproducibility is confirmed across users and runs. This validation framework ensures high specificity, sensitivity, and reproducibility, enabling confident multiomic profiling of complex tissue samples and accelerating translational insights in cancer biology.
利益披露 Disclosure
G. Ong, None..
T. C. Theisen, None..
B. Filanoski, None..
L. Hamanishi, None..
E. Piazza, None..
C. Bailey, None..
P. Divakar, None..
M. L. Hoang, None..
J. M. Beechem, None.