Recombinant Ab markers for stem cell differentiation

描述（由申请人提供）：干细胞疗法的潜力，使一些最普遍的退行性疾病的老年人受益，使其成为一个激烈的研究重点领域。因此，需要相应的试剂来对干细胞及其分化的后代进行分子表征。通过将荧光报告蛋白结合到细胞系特异性基因中进行遗传标记的人胚胎干细胞系将被用作重组噬菌体抗体的诱饵，这些噬菌体抗体可与沿着标记的细胞系进展的细胞特异性相互作用。将采用流式细胞仪进行阳性-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。质谱将被用作鉴定同源抗原的主要方法。此外，试剂还将在ELISA、Western blotting和固定细胞荧光原位检测中发挥作用。确定了第一阶段的两个目标：产生抗体试剂，允许检测特定的细胞系在人类胚胎干细胞分化。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，从高多样性的scFv噬菌体展示文库中选择独特分化阶段的特异性结合物来完成。目标2。鉴定目标1中鉴定的抗体试剂，以确定目标抗原并表征抗体的结合特性。靶标鉴定将通过靶抗原的免疫亲和富集结合MS/MS质谱完成，并通过Western blotting和免疫沉淀等标准生化技术进行确认。（美国国立卫生研究院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.)