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.

中文翻译：

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金属同位素标记的单克隆抗体，用于高维大规模细胞计数。

单细胞大规模细胞计数技术的进步已日益改善了免疫细胞异质性的高度多维表征。免疫测定的多重能力依赖于标记有稳定重金属同位素的单克隆抗体。迄今为止，在质谱分析中已经使用了各种稀土元素以及贵金属和过渡金属同位素。但是，由于单个的金属配位化学以及各种金属阳离子的不同稳定性，用于抗体偶联的方法有所不同。在此，我们提供了三种优化的方案，可将单克隆IgG抗体与48种高纯度重金属同位素结合在一起：（i）镧系元素38种同位素，铟2种同位素和钇1种同位素；（ii）钯的6种同位素；（iii）1个铋的同位素。含有单体DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）或聚合戊酸（DTPA）的配位体的双功能螯合剂用于稳定螯合水溶液中的同位素阳离子，随后使用位点特异性双正交反应与IgG抗体偶联。此外，据报道，根据用二辛可宁酸（BCA）染色后在280 nm处的抗体固有吸收和在562 nm处的外在吸收的定量方法，可以测定标记有金属同位素的抗体。另外，描述了制备钯同位素质量标签的冷冻干燥程序。为了证明其实用性，本文描述了使用六种带有钯标签的CD45抗体通过飞行时间（CyTOF）在细胞计数法中对活免疫细胞进行条形码分析的实验。镧系元素，铟或钇的纯同位素共轭需要约3.5小时。铋的共轭作用需要约4小时。钯质量标签的制备大约需要8个小时。钯的纯同位素共轭需要约2.5小时。抗体滴定需要约4小时。