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

Abstract

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 Reprints and permissions information is available at www.nature.com/reprints.

Beyond the message: advantages of snapshot proteomics with single‐cell mass cytometry in solid tumors

“…Although a large majority of the elemental isotopes used to conjugate antibodies are lanthanides, other metals such as indium, yttrium, and bismuth can also be used. See protocol for conjugating antibodies with these heavy‐metals. Beyond this, users can employ quantum dot labeled antibodies to gain additional channels , iodine for cell cycle tracking , and palladium and platinum for cellular barcoding, allowing advanced users to exceed 50 measurement channels per cell.…”

“…Although a large majority of the elemental isotopes used to conjugate antibodies are lanthanides, other metals such as indium, yttrium, and bismuth can also be used. See protocol [46] for conjugating antibodies with these heavy-metals. Beyond this, users can employ quantum dot labeled antibodies to gain additional channels [47], iodine for cell cycle tracking [124], RNA transcripts [131][132][133], platinum drug uptake [128,129], cell cycle status and DNA synthesis [130], cell size [125], apoptosis [48], and viability [27] Proteins, phospho-proteins, chromatin modifications [145], RNA transcripts [146], fluorescent drug uptake [147], metabolism and redox state [148,149], cell cycle status and DNA synthesis [150], cell size and granularity, apoptosis [151] and viability [152] Poly-adenylated RNA transcripts, CITE-seq probes [144] Minimum cells per sample needed at start of protocol Max is an example maximum range of the instrument scale; error is the typical error for that scale.…”

The end of omics? High dimensional single cell analysis in precision medicine

“…Antibodies labeled with stable metal isotopes prevents spectral overlap issues (albeit some level of compensation might be still required ). Limitations in labeling chemistry restrict today the amount of parameters measured per cell to up to 50 to date , which is still sufficient to apply barcoding strategies (crucial in mass cytometry for the exclusion of doublets and reducing inter‐experimental variability) while preserving high‐sensitivity detection channels . Importantly, once combined, all samples are stained and acquired simultaneously, virtually eliminating technical sample to sample variations and batch effects.…”

Stabilizing Antibody Cocktails for Mass Cytometry

“…In contrast to conventional flow cytometry, where in clinical diagnostic routine readymade liquid antibody cocktails and dry formulations are employed to prevent cocktail pipetting errors , validation data for a comparable procedure for mass cytometry are lacking. Since the vast majority of antibody conjugates for mass cytometry are not covalently associated but rely on chelation of metal ions, as it is the case for lanthanide‐ and palladium‐isotopes , appropriate stabilization and storage conditions have to be established. Although metal chelates used in mass cytometry are characterized by high stability constants (log K > 20) , conferring sufficiently stable association between metal and antibody for some hours or days, and individual antibody conjugates are also stable for months or even years at 4°C if kept isolated from each other, mass‐tagged antibodies have been found to produce unexpected staining patterns when kept mixed together as a cocktail for extended periods of time .…”