质量细胞仪
化学
钯
同位素
单克隆抗体
钇
抗体
生物化学
生物
有机化学
物理
氧化物
免疫学
催化作用
表型
基因
量子力学
作者
Guojun Han,Matthew H. Spitzer,Sean C. Bendall,Wendy J. Fantl,Garry P. Nolan
出处
期刊:Nature Protocols
[Springer Nature]
日期:2018-09-26
卷期号:13 (10): 2121-2148
被引量:168
标识
DOI:10.1038/s41596-018-0016-7
摘要
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. The immunoassay multiplexing capacity of single-cell mass cytometry relies on monoclonal antibodies labeled with stable heavy-metal isotopes. Nolan et al. describe procedures for conjugating monoclonal IgG antibodies with 48 heavy-metal isotopes.
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