Ultrasensitive multiplexed mass cytometry immunoassays based on single patch nanoparticles

本提案描述了无机纳米颗粒(NPs)的合成及其与单抗的偶联研究，特别是含镧系纳米颗粒。NP抗体复合体被设计用于生物标志物和低拷贝数转录本的定量细胞免疫分析。在逐个细胞的基础上分析低拷贝数的生物标志物是细胞生物学的基本兴趣，并为癌症等基于细胞的疾病提供了一种重要的新诊断方法。具有NP抗体结合物的生物检测利用了质量细胞术的多重能力，这是里士满的DVS Sciences在原子质谱仪和细胞学相结合的基础上开发的一种新技术。目前使用DVS质量细胞仪的方法能够使用携带~200个稀土同位素离子的抗体标签来检测每个细胞~2000个水平的生物标记物的表达。为了获得检测每细胞100个拷贝的生物标志物所需的高灵敏度，纳米颗粒必须包含至少5000个单一金属同位素的原子。这需要直径在10到15纳米之间的纳米粒子，其大小与抗体本身相当。这项提议探索了连接稀土纳米颗粒抗体的新的合成策略。对于定量质量细胞术，每个针对特定生物标记物的抗体都应该与单独的NP结合。为了确保抗体不会使抗原发生交联，我们将探索将反应性官能团限制在NPs表面的一小块斑块上的可能性，以确保只有少量(1-4)抗体附着到每个无机纳米颗粒上。金属NP结合物将通过质量细胞仪进行评估，用于量化白血病细胞表面高和低表面生物标记物的表达。然后我们将量化细胞内的抗原，如癌蛋白P53，它在不同的水平上表达，极大地影响细胞生理学。一旦实现，这些NP抗体结合物将用于诊断、监测，并有望找到治疗复杂人类疾病的方法，如癌症、病毒感染如艾滋病、代谢紊乱和心脏病。

This proposal describes research on the synthesis of inorganic nanoparticles (NPs), particularly lanthanide-containing nanoparticles, and their conjugation to monoclonal antibodies. The NP-antibody complexes are designed for quantitative cellular immunoassays of biomarkers and transcripts of low copy number. The analysis of low copy number biomarkers on a cell-by-cell basis is of fundamental interest in cell biology and provides an important new diagnostic for cell based diseases such as cancer. Bioassays with NP-antibody conjugates take advantage of the multiplexing capabilities of mass cytometry, a new technique developed by DVS Sciences, Richmond Hill ON, based upon the union of atomic mass spectrometry and cytometry. Current methods using the DVS mass cytometer are able to detect biomarker expression at the level of ~2000 per cell using antibody tags carrying ~200 ions of a lanthanide isotope. To achieve the high sensitivity needed to detect biomarkers at the level of <100 copies per cell, the nanoparticles must contain atleast 5000 atoms of a single metal isotope. This requires NPs with diameters of 10 to 15 nm, comparable in size to the antibodies themselves. This proposal explores new synthetic strategies for conjugating antibodies to lanthanide nanoparticles. For quantitative mass cytometry, each antibody against a specific biomarker should be bound by a separate NP. To ensure the antibodies do not crosslink the antigens we will explore the potential of confining reactive functional groups to a small patch on the surface of the NPs, with the goal of ensuring that only a small number (1-4) of antibodies will be attached to each inorganic nanoparticle. Metal NP conjugates will be evaluated by mass cytometry for use in quantifying both high and low surface biomarker expression on leukemia cells. Then we will quantify intracellular antigens such as the oncoprotein p53 which is expressed at variable levels dramatically affecting cell physiology. Once realized these NP-antibody conjugates will be useful for the diagnosis, monitoring and hopefully, to find cures for complex human diseases such as cancer, viral infections such as AIDS, metabolic disorders, and heart disease.