Dual-Purpose Polymer Labels for Fluorescent and Mass Cytometric Affinity Bioassays

We describe the synthesis and characterization of a family of poly(N-alkylacrylamide) polymers carrying 2-6 fluorescent dye molecules, ∼70 pendant DTPA (diethylenetriaminepentaacetic acid) groups, and an orthogonal maleimide end-group for covalent attachment to an antibody (Ab). These dual-purpose labels were designed for use in multiplexed immunoassays based on both mass cytometry and fluorescent flow cytometry. A challenge in the polymer synthesis was finding conditions for attaching a sufficient number of dye molecules to each polymer chain. Although attachment of a terminal maleimide to the polymers was not as efficient as anticipated, the end-functional polymers were still effective in labeling Abs. Secondary goat antimouse IgG was labeled with the four dual-label polymers as well as a control polymer, and while the resultant antibody-polymer conjugates showed positive performance in mass cytometric and fluorescent assays, some trials showed problems such as low signal and nonspecific adsorption. Four primary antibody conjugates were prepared and used to stain cells in 4-plex assays. The results of both primary assays are bittersweet in that the CD3-FITC and CD45-DyLight 649 conjugates performed well, while the CD13-DyLight 405 and the CD38-DyLight 549 conjugates did not.