RBC typing antibodies labeled with rare earth phosphor upconverting nanocrystals

描述(由申请人提供)：Sunstone Biosciences和宾夕法尼亚大学提出的STTR申请将产生的产品是一种新型标记抗体试剂盒，可用于在输血前快速识别不同的红细胞表型。目前，输血前配型只涉及ABO和Rh血型的主要抗原，主要依赖于人工血凝反应。这一过程耗时费力，限制了输血前可检测的RBC亚群的数量，有可能使终生接受多次输血的患者产生过敏反应。我们计划开发一大批RBC抗原特异性单抗，用稀土纳米磷标记，能够使用一系列常见和稀有的抗原，以自动化、多重分析的形式快速确定RBC表型。稀土晶体的可编程窄光谱是产生分子检测系统的理想选择，这种系统能够比传统的光学探针具有更大的多路复用能力。在拟议计划的第二阶段结束时，我们预计已经设计和构建了一个由大约20个或更多抗体组成的小组，每个抗体都有唯一的光学发射信号，可以用来创造革命性的血型“特征”。纳米晶体的上转换特性是靶标标记和定量的唯一识别符。其他分子检测方法，包括量子点和传统的荧光团，在整个研究界得到了广泛的应用，但存在许多限制，如光漂白、分辨率差以及允许同时运行的样品数量有限等。稀土荧光粉纳米晶体解决了这些问题，作为惰性、稳定的发光体，允许高分辨率和巨大的多路复用成像。与公共健康相关：太阳石生物科学公司与其在普林斯顿大学的联盟合作伙伴已经产生了初步证据，表明可以合成小至~10 nm的纳米晶体，这些纳米晶体保持其独特的光学性质，并且它们可以被涂层以实现功能化(例如用于与抗体的偶联)。在这个第一阶段的提案中，我们将扩展这些研究，以产生和测试至少三种针对RBC抗原的荧光纳米晶体标记抗体。这项建议将通过开发一种全面匹配供受者血型的新产品，减少输血的过敏反应和其他副作用的发生率。与传统的血液分型方法不同，该方法将允许快速检测即使是稀有的血液标志物。

DESCRIPTION (provided by applicant): The product that will result from the proposed STTR application from Sunstone Biosciences and the University of Pennsylvania is a novel labeled antibody-based reagent kit that can be used to rapidly identify diverse red blood cell phenotypes prior to blood transfusion. At present, pre-transfusion blood typing involves only the major antigens of the ABO and Rh groups, and relies on the use of a largely- manual hemagglutination reaction. The time- and labor-intensiveness of this procedure limits the number of RBC sub-groups that can be tested prior to a transfusion, risking the development of sensitization of patients who receive multiple transfusions throughout their life. We plan to develop a large panel of RBC antigen-specific MAbs, labeled with rare-earth nanophosphors, able to rapidly determine RBC phenotype using a wide range of common and rare antigens in an automatable, multiplexed assay format. The programmable, and narrow optical spectra of rare-earth crystals are ideal for generating molecular detection systems that are capable of vastly greater multiplexing than conventional optical probes. At the end of Phase 2 of the proposed program, we anticipate having designed and constructed a panel of approximately 20 or more antibodies, each with a unique optical emission signal, that can be used to create a revolutionary blood phenotyping "signature". The upconverting characteristics of the nanocrystals are unique identifiers of target-specific labeling and quantification. Other methods of molecular detection, including Quantum Dots and conventional fluorophores, are utilized widely throughout the research community but possess many limitations such as photobleaching, poor resolution, and the limited number of samples permitted to run simultaneously. Rare-earth phosphor nanocyrstals address these problems, as inert, stable light emitters that allow for high resolution and vastly multiplexed imaging. PUBLIC HEALTH RELEVANCE: Sunstones Biosciences has, with its consortium partners at Princeton University, generated preliminary evidence that nanocrystals as small as ~10 nm can be synthesized, that these nanocrystals retain their unique optical properties, and that they can be coated for functionalization (such as for conjugation to antibodies). In this Phase 1 proposal, we will extend these studies to generate and test at least three phosphor nanocrystal-labeled antibodies specific for RBC antigens. This proposal will reduce the incidence of allergic reactions and other side-effects of blood transfusion by developing a new product for comprehensively matching donor and recipient blood types. Unlike conventional methods for blood typing, the proposed method will allow rapid detection of even rare blood markers.