Large scale generation of anti-zebrafish monoclonal antibodies

描述（由申请人提供）：该提案概述了如何将斑马鱼细胞表面和分泌蛋白的独特资源与一种新的抗体选择方法结合使用，以创建200种新的斑马鱼单克隆抗体试剂资源。抗体靶点主要是从它们已知的原位杂交模式中精心选择的，以便对斑马鱼群落有最大的好处。靶点包括：新的细胞标记物和亚细胞定位对功能有重要意义的蛋白质，如形态因子、极性蛋白和神经细胞表面蛋白。选择大量抗体所需的吞吐量将通过使用一种新的池免疫策略、使用蛋白质微阵列的抗体选择和抗体v区的重组克隆来实现。抗体选择过程将识别与来自同一家族的蛋白质无交叉反应的抗体，并识别耐福尔马林表位。最后，每个抗体将在整个胚胎和冷冻切片上进行测试，并将免费和广泛分发。当这些抗体与斑马鱼研究人员可利用的大量遗传资源结合使用时，将允许对蛋白质功能进行新的机制洞察，最终可能用于设计治疗干预的新策略。

DESCRIPTION (provided by applicant): This proposal outlines how a unique resource of zebrafish cell surface and secreted proteins will be used in conjunction with a novel method of antibody selection to create a resource of 200 new zebrafish monoclonal antibody reagents. The antibody targets have been carefully selected primarily from their known in situ hybridization patterns so as to be of maximum benefit to the zebrafish community. Targets include: new cellular markers and proteins whose subcellular localization is important for function such as morphogens, polarity proteins and neural cell surface proteins. The throughput required to select this large number of antibodies will be achieved by using a novel pooling immunization strategy, antibody selection using protein microarrays and the recombinant cloning of antibody V-regions. The antibody selection process will identify antibodies that are non-crossreactive with proteins from the same family and recognize formalin-resistant epitopes. Finally, each antibody will be tested on whole mount embryos and cryosections and will be freely and widely distributed. These antibodies, when used in conjunction with the large genetic resources available to zebrafish researchers, will allow new mechanistic insights into protein function which may eventually be used to design novel strategies for therapeutic intervention. PUBLIC HEALTH RELEVANCE: The zebrafish is an increasingly popular laboratory organism with many experimental advantages that biologists exploit to discover the detailed processes that underlie vertebrate development and disease. Zebrafish research, however, is limited by a paucity of good antibody reagents that are essential tools for determining the precise details of biological processes. This research proposal is aimed at providing the zebrafish community with a major resource of new antibody reagents.