Pichaandi Jothirmayanantham, Zhao Guangyao, Bouzekri Alexandre, Lu Elsa, Ornatsky Olga, Baranov Vladimir, Nitz Mark, Winnik Mitchell A
Department of Chemistry , University of Toronto , 80 St George Street , Toronto , Ontario M5S 3H6 , Canada . Email:
Fluidigm Canada Inc. , 1380 Rodick Road , Markham , Ontario L3R 4G5 , Canada.
Chem Sci. 2019 Jan 23;10(10):2965-2974. doi: 10.1039/c8sc04407d. eCollection 2019 Mar 14.
Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells. In MC, antibodies (Abs) are tagged with heavy metal isotopes conjugation to metal chelating polymers (MCPs). To improve the sensitivity of MC towards low abundance biomarkers, we are developing nanoparticle (NP)-based reagents as mass tags for Abs. We examine the use of silica-coated NaHoF NPs ( ∼ 12 nm) decorated with PEG5k conjugated to thiol-modified primary or secondary Abs for MC assays. We compare the sensitivity of NP-Ab conjugates to MCP-Ab conjugates towards seven biomarkers with varying expression levels across six cell lines. We also perform a multi-parameter assay using a cocktail of both NP- and MCP-based reagents to detect seven cellular markers in peripheral blood mononuclear cells (PBMCs). In the case of highly abundant markers, signal enhancements from NP-Ab conjugates offer minimal advantages over MCP-Ab conjugates, which already give strong signals. In the case of biomarkers with lower abundance, the level of signal enhancements depended on the nature of the biomarker being detected, or on the type of detection method used. When comparing the indirect detection of CD14 on THP-1 cells using NPs or MCPs conjugated to secondary Abs, the NP reagents offered little signal enhancements compared to the MCP reagents. However, in the case of direct CD14 detection on THP-1 or U937 cells using NPs or MCPs conjugated to primary Abs, a 30- or 450-fold signal enhancement was seen from the NP-based reagent. In the experiments where both NP-Ab and MCP-Ab conjugates were used together to stain PBMCs, we found that the presence of the NP-Ab conjugates did not affect the function of MCP-Ab conjugates, and the NP-Ab conjugates showed minimal non-specific interaction with cells without the target biomarker (CD14). Furthermore, these NP-Ab conjugates could be used to identify rare CD14 monocytes from the PBMC mixture with a 20-fold signal increase when compared to the use of only MCP-Ab conjugates. Collectively, the strong signal amplification obtained from NP reagents demonstrate the potential of these reagents to be used in conjunction with MCP-reagents to detect rare cellular markers or cell types that may otherwise be overlooked when using MCP-reagents alone.
质谱流式细胞术（MC）是一种用于确定细胞中生物标志物表达的高通量多参数分析技术。在MC中，抗体（Abs）用与金属螯合聚合物（MCPs）共轭的重金属同位素标记。为了提高MC对低丰度生物标志物的灵敏度，我们正在开发基于纳米颗粒（NP）的试剂作为Abs的质量标签。我们研究了用与硫醇修饰的一抗或二抗共轭的PEG5k修饰的二氧化硅包覆的NaHoF NPs（~12 nm）用于MC分析。我们比较了NP-Ab共轭物与MCP-Ab共轭物对六种细胞系中七种表达水平不同的生物标志物的灵敏度。我们还使用基于NP和MCP的试剂混合物进行多参数分析，以检测外周血单核细胞（PBMCs）中的七种细胞标志物。对于高丰度标志物，NP-Ab共轭物的信号增强与已经给出强信号的MCP-Ab共轭物相比优势不大。对于丰度较低的生物标志物，信号增强水平取决于所检测生物标志物的性质或所使用的检测方法类型。当比较使用与二抗共轭的NP或MCP对THP-1细胞上CD14的间接检测时，与MCP试剂相比，NP试剂的信号增强很少。然而，在使用与一抗共轭的NP或MCP对THP-1或U937细胞上CD14进行直接检测的情况下，基于NP的试剂的信号增强了30倍或450倍。在同时使用NP-Ab和MCP-Ab共轭物对PBMCs进行染色的实验中，我们发现NP-Ab共轭物的存在不影响MCP-Ab共轭物的功能，并且NP-Ab共轭物与没有目标生物标志物（CD14）的细胞的非特异性相互作用最小。此外，与仅使用MCP-Ab共轭物相比，这些NP-Ab共轭物可用于从PBMC混合物中鉴定罕见的CD14单核细胞，信号增加20倍。总体而言，从NP试剂获得的强信号放大证明了这些试剂与MCP试剂联合使用以检测罕见细胞标志物或细胞类型的潜力，否则单独使用MCP试剂时可能会忽略这些标志物或细胞类型。
