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- W2893573432 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 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." @default.
- W2893573432 created "2018-10-05" @default.
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- W2893573432 creator A5058850325 @default.
- W2893573432 creator A5071972147 @default.
- W2893573432 date "2018-09-26" @default.
- W2893573432 modified "2023-10-18" @default.
- W2893573432 title "Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry" @default.
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- W2893573432 doi "https://doi.org/10.1038/s41596-018-0016-7" @default.
- W2893573432 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7075473" @default.
- W2893573432 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/30258176" @default.
- W2893573432 hasPublicationYear "2018" @default.
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