Llama-derived phage display antibody arrays for FSHD

描述(申请人提供)：本项目的目的是获得洞察力 在细胞和分子过程中导致细胞功能障碍 FSHD患者神经肌肉系统蛋白质的大规模分析 患者和对照组组织和细胞系的动态平衡。 在过去的几年里，已经启动了一些项目，研究放松对 FSHD在RNA水平上的生物学途径。这些策略包括差异化 商业和定制DNA芯片上的展示和RNA图谱实验 和数组。尽管存在局限性，DNA阵列现在是最多的 常用和成功的测定分子和细胞的方法 许多获得性和遗传性疾病的某些方面。预计也将用于 FSHD，这种方法将为理解其 病理学。然而，蛋白质水平，包括修饰的水平 蛋白质和蛋白质复合体的组成具有重要的顺序 了解FSHD的病理生理学。因此，有必要 蛋白质阵列。 骆驼抗体技术为开发蛋白质提供了一个独特的机会 数组。骆驼系统为此目的的力量是，这种动物 制造单(重)链抗体。利用遗传信息实现这一点 构建噬菌体展示抗体的单链抗体库 图书馆不再需要将重链和轻链结合在一起，这是 传统噬菌体展示文库的主要缺点。此外，这些 单链抗体往往具有非常高的亲和力和稳定性。它有 已经证明，大量幼稚和定向的抗体库 可以生成。在克隆、生产和分离这些病毒方面的经验 骆驼抗体是可用的。 我们建议从骆驼中产生肌肉特异性抗体阵列 单链噬菌体展示克隆。为此，骆驼将用 人肌肉蛋白匀浆，峰值反应后，噬菌体展示 图书馆将建成。抗体克隆将用各种不同的 选择程序(例如，使用重组蛋白或肌肉匀浆 来自不同物种)，并排列在玻璃幻灯片上。很好地描述了单一 链状抗体阵列将用于研究FSHD的病理生理学 组织和细胞培养的荧光标记蛋白匀浆 病人和对照组。显然，这些抗体也可以用来 单独用于特定的免疫组织化学和免疫细胞化学研究。

DESCRIPTION (provided by applicant): The aim of this project is to gain insight in the cellular and molecular processes leading to dysfunction of the neuromuscular system in FSHD patients by large scale analysis of protein homeostasis in tissues and cell lines of patients and controls. In the past few years, projects have been launched to study deregulation of biological pathways in FSHD on RNA level. These strategies include differential display and RNA profiling experiments on commercial and custom made DNA chips and arrays. Despite their limitations, DNA arrays are now one of the most commonly used and successful methods to determine the molecular and cellular aspects of many acquired and genetic diseases. It is anticipated that also for FSHD, this approach will provide a valuable contribution in understanding its pathology. Nevertheless, protein levels, including the level of modified proteins and the composition of protein complexes are of an order of importance larger to understand FSHD pathophysiology. Consequently there is a need for protein arrays. The llama antibody technology provides a unique opportunity to develop protein arrays. The power of the llama system for this purpose is that this animal makes single (heavy) chain antibodies. Using the genetic information for this single-chain repertoire for the construction of phage-display antibody libraries abolishes the need to combine heavy- and light-chains, one of the major drawbacks of conventional phage-display libraries. Moreover, these single-chain antibodies tend to have a very high affinity and stability. It has already been demonstrated that large naive and directed libraries of antibodies can be generated. Experience in cloning, production and isolation of these llama antibodies is available. We propose to generate muscle-specific antibody arrays derived from Llama single-chain phage-display clones. To this end, a Llama will be immunized with human muscle protein homogenates, and after peak response, a phage display library will be constructed. Antibody clones will be selected with a variety of selection procedures (e.g. with recombinant proteins or with muscle homogenates from different species) and arrayed on glass slides. Well characterized single chain antibody arrays will be used to study FSHD pathophysiology on fluorescently labeled protein homogenates of tissues and cell cultures of patients and controls. Evidently, these antibodies can also be used individually for specific immunohistochemical and immunocytochemical studies.