Measuring the Degree of Labeling of Antibody-Dye Conjugates with a Single-Molecule-Sensitive Digital Flow Cytometer

Antibody-dye conjugates are an essential tool in biomedical research and are widely used in flow cytometry, immunofluorescence imaging, and ELISAs. The degree of labeling (DOL), often referred to as the F/P (fluorophore-to-protein) ratio, which is the number of dyes per antibody, is a fundamental characteristic of antibody-dye conjugates that impacts conjugate performance (emission intensity, affinity, solubility/aggregation) and the accuracy and reproducibility of results. Knowing the DOL of antibody-dye conjugates is essential for accurately quantifying target proteins and/or antigens in quantitative flow cytometry and quantitative immunofluorescence. In CAR-T cell therapy, the expression level of the chimeric antigen receptor (CAR) on T cells must be finely tuned to optimize treatment efficacy but cannot be calculated without the DOL of the anti-CAR antibody-dye conjugate. However, DOL cannot be easily measured (e.g., via absorption spectroscopy) for many widely used conjugates that contain protein-based or semiconducting polymer/nanoparticle dyes due to overlap in absorption cross-section between antibody and dye. Bulk analysis also cannot reveal the distribution of DOL values, which can impact conjugate performance and biomarker quantitation. Furthermore, quenching between dyes can reduce the conjugate emission. Herein, we demonstrate the use of a single-molecule-sensitive digital flow cytometer (dFC) with 100% single-fluorophore detection efficiency to measure the fluorescence emission distributions of conjugates and free dyes and perform deconvolution to obtain effective DOL values (the apparent number of dyes per conjugate based on fluorescence emission, useful for converting fluorescence intensity to antigens/cell) for antibodies conjugated with small-molecule, protein-based, and semiconducting polymer/nanoparticle dyes.