Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK.
BMC Biol. 2010 Jun 4;8:76. doi: 10.1186/1741-7007-8-76.
Monoclonal antibodies with high affinity and selectivity that work on wholemount fixed tissues are valuable reagents to the cell and developmental biologist, and yet isolating them remains a long and unpredictable process. Here we report a rapid and scalable method to select and express recombinant mouse monoclonal antibodies that are essentially equivalent to those secreted by parental IgG-isotype hybridomas.
Increased throughput was achieved by immunizing mice with pools of antigens and cloning - from small numbers of hybridoma cells - the functionally rearranged light and heavy chains into a single expression plasmid. By immunizing with the ectodomains of zebrafish cell surface receptor proteins expressed in mammalian cells and screening for formalin-resistant epitopes, we selected antibodies that gave expected staining patterns on wholemount fixed zebrafish embryos.
This method can be used to quickly select several high quality monoclonal antibodies from a single immunized mouse and facilitates their distribution using plasmids.
对全固定组织具有高亲和力和选择性的单克隆抗体是细胞和发育生物学家的宝贵试剂，但分离它们仍然是一个漫长而不可预测的过程。在这里，我们报告了一种快速且可扩展的方法来选择和表达重组小鼠单克隆抗体，这些抗体在本质上与亲本 IgG 同型杂交瘤分泌的抗体相当。
通过用抗原池免疫小鼠，并从小数目的杂交瘤细胞中克隆功能重排的轻链和重链到单个表达质粒中，提高了通量。通过用在哺乳动物细胞中表达的斑马鱼细胞表面受体蛋白的细胞外结构域免疫，并筛选福尔马林抗性表位，我们选择了在全固定斑马鱼胚胎上给出预期染色模式的抗体。
该方法可用于从单个免疫小鼠中快速选择几种高质量的单克隆抗体，并使用质粒方便地进行其分配。
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