Llama-derived phage display antibody arrays for FSHD

用于 FSHD 的源自美洲驼的噬菌体展示抗体阵列

• 批准号: 6534543
• 负责人: SILVERE M VAN DER MAAREL
• 金额: $ 12.5万
• 依托单位: LEIDEN UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534543
• 关键词: Ungulate; antibody; bacterial virus; complementary DNA; genetic library; genetically modified animals; histopathology; human subject; immunofluorescence technique; laboratory mouse; microarray technology; muscle proteins; muscular dystrophy; pathologic process; patient oriented research; technology /technique development; western blottings

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.

描述（由申请人提供）：该项目的目的是获得洞察力 在细胞和分子过程中导致功能障碍 通过大规模蛋白质分析研究 FSHD 患者的神经肌肉系统 患者和对照的组织和细胞系的稳态。 过去几年，已经启动了研究放松管制的项目 FSHD 在 RNA 水平上的生物学途径。这些策略包括差异化 在商业和定制 DNA 芯片上进行显示和 RNA 分析实验 和数组。尽管存在局限性，DNA 阵列现在仍是最先进的阵列之一 确定分子和细胞的常用且成功的方法 许多获得性和遗传性疾病的方面。预计也将用于 FSHD，这种方法将为理解其 病理。然而，蛋白质水平，包括修饰的水平 蛋白质和蛋白质复合物的组成具有一定的重要性 更大程度地了解 FSHD 病理生理学。因此需要 蛋白质阵列。 美洲驼抗体技术提供了开发蛋白质的独特机会 数组。美洲驼系统为此目的的力量在于，这种动物 产生单（重）链抗体。为此使用遗传信息 用于构建噬菌体展示抗体的单链库 文库消除了组合重链和轻链的需要，这是 传统噬菌体展示文库的主要缺点。而且，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，大型天然和定向抗体库 可以生成。具有克隆、生产和分离这些物质的经验 可获得美洲驼抗体。 我们建议生成源自美洲驼的肌肉特异性抗体阵列 单链噬菌体展示克隆。为此，将对美洲驼进行免疫接种 人类肌肉蛋白匀浆，峰值反应后，噬菌体展示 将建设图书馆。抗体克隆将通过多种选择 选择程序（例如使用重组蛋白或肌肉匀浆 来自不同物种）并排列在载玻片上。性格鲜明的单身 链抗体阵列将用于研究 FSHD 病理生理学 组织和细胞培养物的荧光标记蛋白匀浆 患者和对照。显然，这些抗体也可以使用 单独用于特定的免疫组织化学和免疫细胞化学研究。