Multi-step antibody validation for nCounter high-plex multiomics panels

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.