Antibody Core

摘要 负责抗体工程和生产的核心将服务于#4项目，以及#1和#3核心。 将生产和分发多克隆抗体和单克隆抗体。工作流程将被组织起来 沿着4项具体任务的路线。任务1：制备和鉴定抗多聚糖多克隆和单抗 抗体。免疫后，我们将使用两种主要方法来产生抗体：标准融合到 一对永生配对产生单抗的经典方式，并进行重组配对表达 单B细胞抗体链测序后CHO细胞中的重(H)链和轻(L)链(由Project提供 #3)。当需要大量多克隆抗体时，将通过免疫大量的 动物和定期出血。单抗将被亚克隆和同型。经过提纯后， 感兴趣的抗体将被表征为通过表面等离子体共振结合到目标糖链上 Biacore T200。重组免疫球蛋白A和免疫球蛋白M将在昆虫细胞中产生。 任务2：生产用于结构研究和其他方面的抗体的Fab片段。结晶很大程度上 通过去除抗体的Fc片段和产生Fab片段来促进。制作 这些片段将通过两条途径实现。第一种是全长的蛋白水解性切割。 抗体与木瓜酶等酶结合，第二种是瞬时重组表达的配对 CHO细胞中截短的H-全长L链。 任务3：修改FC片段以修改效应器功能。黄曲霉毒素的抗菌作用 抗体高度依赖于抗体的Fc片段相关功能。FC组分 不仅决定半衰期，而且还决定补体的结合和激活，以及与一些细菌的结合 蛋白质，如蛋白质A，小鼠和人类的Fc片段都将被嫁接和/或突变，以改变 一种特定抗体的生物学活性并在CHO细胞重组表达系统中表达。 任务4：质量控制、储存和配送。这一特定目标对于一个核心的成功至关重要。 在一笔大笔合作拨款内。所有试剂的电子存档将集中在一台计算机上 并备份在单独的硬盘驱动器和机构备份系统上。对于每一种抗体 兴趣、免疫原特性、融合日期、同种异型、结合常数将在母版中介绍 电子表格。H和L序列将可用于单细胞测序重新表达的抗体。 DNA和细胞将分别储存在零下80摄氏度和液氮中。完整描述的抗体列表如下 可在PO1的网站上查阅，并定期更新。集团内的分配将是 在我们的月度会议上讨论过。外部分销将遵循MTA协议的制度规则。

Abstract The core in charge of antibody engineering and production will serve projects #4, as well as Cores #1 and #3. Polyclonal as well as monoclonal antibodies will be produced and distributed. The flow of work will be organized along the lines of 4 specific tasks. Task 1: Produce and characterize anti-glycan polyclonal and monoclonal antibodies. Following immunization, we will use two main approaches to produce antibodies: standard fusion to an immortal partner to produce monoclonal antibodies the “classical” way, and expression of recombinant paired Heavy (H) and Light (L) chains in CHO cells after single B cell antibody chain sequencing (provided by Project #3). Polyclonal antibodies when needed in large quantities will be produced by immunizing large number of animals and regular bleeding. Monoclonal antibodies will be subcloned and isotyped. After purification, antibodies of interest will be characterized for binding to the target glycan by surface plasmon resonance on a Biacore T200. Recombinant IgA and IgM will be produced in insect cells. Task 2: Produce Fab fragments of antibodies for structural studies and others. Crystallization is greatly facilitated by the removal of the Fc fragment of antibodies and the production of Fab fragments. The production of these fragments will be accomplished by two routes. The first one is the proteolytic cleavage of the full-length antibodies with enzymes such as papain, the second one is the transient recombinant expression of paired truncated H - full length L chains in CHO cells. Task 3: Modifications of the Fc fragment to modify effector functions. The antibacterial functions of antibodies are highly dependent on the Fc fragment-associated functions of antibodies. Fc composition determines not only half-life but also complement binding and activation, as well as binding to some bacterial protein such as protein A. Both mouse and human Fc fragments will be grafted and/or mutated to modify the bioactivity of a particular antibody and expressed in a CHO cell recombinant expression system. Task 4: Quality control, storage, and distribution. This particular aim is essential to the success of a core within a large collaborative grant. Electronic archiving of all reagents will be centralized on a single computer and backed up on a separate hard drive as well as on the institutional backup system. For each antibody of interest, immunogen characterization, date of fusion, isotype, binding constants will be presented in a master spreadsheet. H and L sequences will be accessible for antibodies re-expressed from single cell sequencing. DNA and cells will be stored at -80ºC and LN2, respectively. The list of the fully described antibodies will be available on the Website of the PO1 and updated on a regular basis. Distribution within the group will be discussed at our monthly meetings. Outside distribution will follow the institutional rules of MTA agreement.