Recombinant Ab markers for stem cell differentiation

描述(由申请人提供)：干细胞疗法的潜力，使患有老龄化人口中一些最普遍的退行性疾病的患者受益，这使这一领域成为研究的重点。相应地，需要能够对干细胞及其分化后代进行分子表征的试剂。在这里，人类胚胎干细胞系通过将荧光报告蛋白掺入细胞谱系特异性基因来进行遗传标记，将被用作重组噬菌体抗体的诱饵，这些重组噬菌体抗体与沿着标记谱系前进的细胞特异性地相互作用。将采用FACS的阳性-阴性选择，允许重复富集步骤，以实现高特异性试剂的分离。质谱学将被用作鉴定同源抗原的主要方法。此外，还将对试剂在酶联免疫吸附试验、Western blotting和固定细胞上的荧光原位检测中的用途进行鉴定。确定了两个阶段1的目标：目标1.产生抗体试剂，允许在人类胚胎干细胞分化过程中检测特定的细胞谱系。这将通过利用荧光报告标记的基因陷阱细胞系和荧光激活的细胞分选，在独特的分化阶段从高度多样性的ScFv噬菌体展示文库中选择特定的结合子来实现。目标2.对目标1中确定的抗体试剂进行表征，以确定目标抗原并表征抗体的结合特性。目标识别将通过目标抗原的免疫亲和浓缩结合MS/MS质谱学完成，并通过标准生化技术如Western blotting和免疫沉淀进行确认。(NIH人类胚胎干细胞注册细胞系UC06将用于拟议的工作。)

DESCRIPTION (provided by applicant): The potential of stem cell therapies to benefit patients afflicted by some of the most pervasive degenerative diseases of an aging population have made this an intense area of research focus. There is a corresponding need for reagents that permit the molecular characterization of stem cells and their differentiated progeny. Here human embryonic stem cell lines which are genetically marked by incorporation of a fluorescent reporter protein into cell lineage specific genes will be used as bait for recombinant phage antibodies that specifically interact with cells progressing along the marked lineage. A positive-negative selection by FACS will be employed allowing repeated enrichment steps to achieve the isolation of highly specific reagents. Mass spectroscopy will be used as a primary approach to identifying the cognate antigens. Reagents will be additionally characterized for their utility in ELISA, Western blotting and fluorescence in situ detection on fixed cells. Two Phase 1 goals are identified: Goal 1. Generation of antibody reagents allowing detection of specific cell lineages during human embryonic stem cell differentiation. This will be accomplished by the selection of specific binders from a high diversity scFv phage display library at unique differentiation stages by taking advantage of fluorescent reporter marked gene trap cell lines and fluorescence activated cell sorting. Goal 2. Characterization of antibody reagents identified in Goal 1 to define the target antigen and characterize the binding properties of the antibodies. Target identification will be accomplished by immunoaffinity enrichment of the target antigen combined with MS/MS mass spectroscopy and confirmed by standard biochemical techniques such as Western blotting and immune precipitation. (The NIH Human Embryonic Stem Cell Registry cell line UC06 will be used in the proposed work.)