Amelie Croset

Among various cellular characteristics, flow cytometry can evaluate antigen expression through qualitative or quantitative approaches. For relative quantification, fluorescence intensity (FI) values are converted into quantitative measurements using appropriate reference materials. To quantitatively estimate antigen density or define ligand‐binding sites per cell, antibody binding capacity (ABC) values serve as the preferred metric. Standardizing assays through the conversion of arbitrary FI units into quantitative data is essential for consistency. However, reported ABC values for well‐characterized antigens vary across the literature. This study addresses the challenges in achieving robust and reproducible quantitative flow cytometry data, offering methodological recommendations for accurately assessing target expression. Our research includes a comprehensive investigation of multiple factors, such as conventional and full‐spectrum instruments, antibodies, reagents, matrices, cell density/confluency, cellular autofluorescence, and quantitative kits, to identify the primary sources of variation in ABC calculations. By implementing a systematic and integrated approach, we aim to ensure the generation of reliable and reproducible ABC values. Longitudinal studies provide strong evidence of assay robustness, while the established protocol further supports biomarker evaluation across different matrices and various stages of drug development.

Supplementary Fig. S3. TAA quantification on healthy blood cells.

Supplementary Fig. S15. TCE-induced CD70 upregulation on activated CD4 and CD8 T cells. Expression of CD70 on T cells in cocultures with MOLM-13 cells and 1 nmol/L MP0533 or CD123-CD3 DART.

Supplementary Fig. S17. TAA expression analysis on AML PDX cells.

Supplementary Fig. S6. Characterization of MOLM-13 clones.

Supplementary Fig. S2. MP0533 affinity traces and simultaneous binding to its targets.

Supplementary Fig. S10. Competition/inhibition with soluble ligands/TAAs.