Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry

• Published in: Nature protocols Vol. 13; no. 10; pp. 2121 - 2148
• Main Authors: Han, Guojun, Spitzer, Matthew H, Bendall, Sean C, Fantl, Wendy J, Nolan, Garry P
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.10.2018
• Subjects: Absorption; Animals; Antibodies, Monoclonal - chemistry; Antigens, CD - analysis; Bismuth; Bismuth - chemistry; Cationic polymerization; Cations; CD45 antigen; Chelating agents; Chelating Agents - chemistry; Chelation; Chemical properties; Conjugation; Cytometry; Flow cytometry; Flow Cytometry - methods; Freeze drying; Heavy metals; Heterocyclic Compounds, 1-Ring - chemistry; Heterogeneity; Humans; Immune system; Immunoassay; Immunoassay - methods; Immunoconjugates - chemistry; Immunoglobulin G; Immunoglobulin G - chemistry; Immunoglobulins; Indium; Indium - chemistry; Isotopes; Isotopes - chemistry; Jurkat Cells; Lanthanides; Metal ions; Metals; Metals - chemistry; Methods; Mice, Inbred C57BL; Monoclonal antibodies; Multiplexing; Organic chemistry; Palladium; Palladium - chemistry; Pentetic acid; Rare earth elements; Rare earth metal compounds; Tags; Titration; Yttrium; Yttrium - chemistry; United States
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/s41596-018-0016-7

Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.