A comprehensive analytical validation framework for immunofluorescence assay design using CellScape precise spatial multiplexing

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Presentation Time: 03:15 PM - 04:30 PM
Poster Board Number: B910
Abstract ID: 5249

Presenting Author:
Oliver Braubach , Director Research and Development at Canopy Biosci.

Abstract:

Multiplex immunofluorescence (mIF) is a powerful immunology research tool that enables deep phenotyping of cells within the tissue microenvironment.  Careful optimization of multiplex antibody panels for specificity, sensitivity, and reproducibility, is crucial to achieving robust data and is a requirement for assay repeatability and standardization. Here, we describe our validation procedure for multiplex assay panels.

Experiments were performed using the CellScape™ platform for precise spatial multiplexing. 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 in a multi-stage testing matrix to validate each antibody in isolation and assembled into a panel. Measurements of both signal and quantitative immune phenotyping across technical replicates showed a high degree of intra-assay precision and inter-assay reproducibility.

Pairing the CellScape platform with comprehensively validated antibody panels streamlined deep immune profiling. The 15-plex, ready-to-use VistaPlex™ immune profiling panel for human FFPE tissues was validated on human tonsil, lung, breast, and colon tissues demonstrating the ability to positively identify epithelial cells and 22 cellular immune phenotypes most critical to understanding tumor immune responses.