Recombinant Ab markers for stem cell differentiation

描述（由申请人提供）：干细胞疗法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的潜力使其成为研究重点的一个激烈领域。相应地需要允许干细胞及其分化后代的分子表征的试剂。在此，通过将荧光报道蛋白掺入细胞谱系特异性基因而遗传标记的人胚胎干细胞系将用作重组噬菌体抗体的诱饵，所述重组噬菌体抗体与沿标记谱系沿着的细胞特异性相互作用。将采用通过FACS进行的阳性-阴性选择，允许重复富集步骤以实现高度特异性试剂的分离。质谱将用作鉴定同源抗原的主要方法。试剂将另外表征其在ELISA、蛋白质印迹和固定细胞上的荧光原位检测中的效用。确定了两个阶段1目标：目标1。在人胚胎干细胞分化过程中产生允许检测特定细胞谱系的抗体试剂。这将通过在独特的分化阶段从高多样性scFv噬菌体展示文库中选择特异性结合剂来实现，所述特异性结合剂通过利用荧光报告标记的基因捕获细胞系和荧光激活的细胞分选来实现。目标2.对目标1中确定的抗体试剂进行表征，以确定靶抗原并表征抗体的结合特性。通过靶抗原的免疫亲和富集结合MS/MS质谱法完成靶标鉴定，并通过标准生物化学技术（如Western印迹和免疫沉淀）进行确认。(The NIH人类胚胎干细胞登记处细胞系UC 06将用于拟定工作。）

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.)