Large scale generation of anti-zebrafish monoclonal antibodies

描述(由申请人提供)：这份建议书概述了如何将斑马鱼细胞表面和分泌蛋白质的独特资源与一种新的抗体选择方法结合使用，以创建200种新的斑马鱼单抗试剂资源。抗体靶标主要是从已知的原位杂交模式中精心挑选出来的，以便对斑马鱼群落产生最大的好处。靶点包括：新的细胞标志物和亚细胞定位对功能重要的蛋白质，如形态原、极性蛋白和神经细胞表面蛋白。选择如此多的抗体所需的吞吐量将通过使用新的混合免疫策略、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同一家族的蛋白质无交叉反应的抗体，并识别福尔马林抗性表位。最后，每种抗体都将在整个胚胎和冷冻切片上进行测试，并将自由和广泛地分布。当这些抗体与斑马鱼研究人员可用的大量遗传资源结合使用时，将允许对蛋白质功能的新的机械性见解，最终可能被用于设计治疗干预的新策略。 与公共健康相关：斑马鱼是一种越来越受欢迎的实验室生物，具有许多实验优势，生物学家利用这些优势来发现脊椎动物发育和疾病背后的详细过程。然而，斑马鱼的研究受到良好抗体试剂匮乏的限制，这些抗体试剂是确定生物过程的确切细节的必要工具。这项研究建议旨在为斑马鱼界提供新的抗体试剂的主要资源。

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.