This thesis describes the synthesis, characterization, and application of metal-chelating polymers for mass-cytometric bioassays. Mass cytometry is a cell characterization technique in which cells are injected individually into an ICP-MS detector. Signal is provided by staining cell-surface or intracellular antigens with metal-labeled antibodies (Abs). These Abs are labeled through the covalent attachment of metal-chelating polymers which carry multiple copies of a lanthanide isotope.
In this work, my first goal was to develop a facile, straightforward synthesis of a new generation of metal-chelating polymers. The synthesis began with reversible addition-fragmentation chain transfer polymerization, and was followed by numerous post-polymerization pendant group transformations to introduce DTPA lanthanide chelators to every repeat unit, and a maleimide at the end of the chain.
The second goal was to apply these metal-chelating polymers in bioassay experiments. The DTPA groups were loaded with lanthanide ions, and the maleimide group was used to covalently attach the polymer to an Ab. This goat anti-mouse conjugate was found to carry an average of 2.4 ± 0.3 polymer chains. Then, primary Ab conjugates were prepared and used in an 11-plex mass cytometry assay in the characterization of umbilical cord blood cells.
The third goal was to expand the multiplexity of the assay. In current technology, the number of Abs that can be monitored simultaneously is limited to the 31 commercially available, stable lanthanide isotopes. Thus, I had an interest in preparing metal-chelating polymers that could carry other metals in the 100-220 amu range. I synthesized polymers with four different polyaminocarboxylate ligands, and investigated the loading of palladium and platinum ions into these polymers. Polymer-Ab conjugates prepared with palladium- and platinum-loaded polymers gave curious results, in that only dead cells were recognized.
The fourth goal was to create dual-purpose Ab tags. My approach was to synthesize polymers similar to those described above, but which also carried two to six fluorescent dyes. Polymer-Ab conjugates prepared with four different dye-labeled polymers gave mixed results. Two of the four conjugates performed well in FACS and mass cytometric assays, but the other two did not. Further experiments are needed to overcome this problem.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/44078 |
| Date | 20 March 2014 |
| Creators | Majonis, Daniel |
| Contributors | Winnik, Mitchell A |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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