Synthesis of a Functional Metal-Chelating Polymer and Steps toward Quantitative Mass Cytometry Bioassays

Abstract

We describe the synthesis and characterization of metal-chelating polymers with a degree of polymerization of 67 and 79, high diethylenetriaminepentaacetic acid (DTPA) functionality, M_w/M_n ≤ 1.17, and a maleimide as an orthogonal functional group for conjugation to antibodies. The polymeric disulfide form of the DP_n = 79 DTPA polymer was analyzed by thermogravimetric analysis to determine moisture and sodium-ion content and by isothermal titration calorimetry (ITC) to determine the Gd³⁺ binding capacity. These results showed each chain binds 68 ± 7 Gd³⁺ per chain. Secondary goat antimouse IgG was covalently labeled with the maleimide form of the DTPA polymer (DP_n = 79) carrying ¹⁵⁹Tb. Conventional ICPMS analysis of this conjugate showed each antibody carried an average of 161 ± 4 ¹⁵⁹Tb atoms. This result was combined with the ITC result to show there are an average of 2.4 ± 0.3 polymer chains attached to each antibody. Eleven monoclonal primary antibodies were labeled with different lanthanide isotopes using the same labeling methodology. Single cell analysis of whole umbilical cord blood stained with a mixture of 11 metal-tagged antibodies was performed by mass cytometry.