Accelerated molecular probe pipeline

描述(由申请人提供)：这笔赠款将开发、验证和利用快速生成分子探针的新方案。分子探针(通常是单抗)的产生仍然是生物医学研究、生物标记物发现和诊断测试开发的关键瓶颈。已经开发了几种方法来克服这个问题。例如，酵母展示文库在酿酒酵母细胞表面表达多种单链片段可变抗体(ScFv)。通过使用荧光激活细胞分选(FACS)，可以在2到3周内从原始文库中挑选出与抗原特异结合的酵母克隆。然而，在酵母细胞表面表现良好的选定探针在溶液中往往表现不佳。为了实现一个更广泛的有用的、更快的分子探针管道，这笔赠款将利用酵母单链抗体展示的速度和吞吐量，同时克服其固有的限制。酵母免疫沉淀-串联质谱学(酵母IP-MS/MS)方法将被用于提高生物标志物研究的速度和吞吐量。新型纳米粒子支架与流式细胞仪的结合将被用于提高单链抗体与抗原结合的功能亲和力。作为开发这些方法的典范，该项目将确定溶组织内阿米巴的新的包囊抗原，这是一种重要的肠道病原体和B类病原体。加速分子探针流水线(AMPP)将首先用于鉴定和验证这些生物标志物，然后产生高活性的单链抗体探针，用于改进的粪便溶组性肠杆菌包囊诊断试验。相关性(参见说明)：如果成功，该项目将改变分子探针的生成方式，用于研究、生物标记物发现和诊断测试。它将有利于任何利用分子探针的研究或开发工作，并为新的疫苗、药物和诊断铺平更快的道路。

DESCRIPTION (provided by applicant): This grant will develop, validate, and utilize novel protocols for rapid generation of molecular probes. The generation of molecular probes (usually monoclonal antibodies) remains a critical bottleneck in biomedical research, biomarker discovery, and diagnostic test development. Several approaches have been developed to overcome this problem. For example, yeast display libraries express diverse single chain-fragment variable (scFv) antibodies on the surfaces of Saccharomyces cerevisiae cells. By using fluorescent-activated cell sorting (FACS), yeast clones that bind specifically to antigens can be selected from naive libraries in 2 to 3 weeks. However, selected probes that perform well on yeast cell surfaces often perform poorly in solution. In order to achieve a more broadly useful, accelerated pipeline for molecular probes, this grant will leverage the speed and throughput of yeast scFv display, while overcoming its innate limitations. A yeast immunoprecipitation - tandem mass spectroscopy (yeast IP-MS/MS) approach will be used to improve the speed and throughput of biomarker research. Novel nanoparticle scaffolds combined with FACS will be used to improve the functional affinity of scFv binding to antigens. As a model for the development of these approaches, the project will identify novel cyst antigens of Entamoaeba histolytica, an important enteric pathogen and Category B agent. The accelerated molecular probe pipeline (AMPP) will be used first to identify and validate these biomarkers, and then to generate high-activity scFv probes for incorporation into an improved diagnostic test for E. histolytica cysts in stool. RELEVANCE (See instructions): If successful, this project will change the way molecular probes are generated for research, biomarker discovery, and diagnostic testing. It will benefit any research or development effort that utilizes molecular probes, and pave faster routes to new vaccines, drugs, and diagnostics.