Abstract 1539: A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay

Abstract Background: Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of cells within the tumor microenvironment (TME). Careful optimization of multiplex antibody panels for specificity, sensitivity and reproducibility is crucial to achieving robust data and is a precondition for assay repeatability and standardization. We describe our validation procedure for VistaPlex™ multiplex assay panels. Methods: Experiments were performed using the CellScape™ platform for precise spatial multiplexing. The platform enables automated cyclic mIF for readily available fluorophore-conjugated antibodies compatible with fresh frozen or FFPE tissues. The use of high dynamic range imaging further facilitates quantitative phenotyping, while excellent imaging resolution provides single-cell discrimination with high accuracy. To design a ready-to-use panel for immune cell phenotyping, individual antibodies were first screened for specificity and then optimized for labeling sensitivity. Signal-to-noise, inter-assay reproducibility, and intra-assay precision were evaluated across serial sections in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Resulting from this effort, we introduce a 15-plex ready-to-use VistaPlex immune profiling panel for human FFPE tissues. Results: Image analysis of signal-to-noise ratios demonstrated robust performance of multiplexed antibodies across multiple tissue sections and tissue types. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility. The 15-plex panel was validated on human tonsil, lung, breast, and colon tissues. Conclusions: The CellScape platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Rigorously validated VistaPlex panels further streamline this spatial biology workflow and will aid in the generation of consistent and reliable results for end users. Citation Format: Charles E. Jackson, Arne Christians, Jannik Boog, Matthew H. Ingalls, Xenia Meshik, Adam Northcutt, Kayla E. Cashion, J Spencer Schwarz, Oliver Braubach. A comprehensive analytical validation framework for antibodies used in the CellScape multiplex immunofluorescence assay abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1539.