UCD Centre for Nanomedicine, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland.
Nat Protoc. 2013 Jun;8(6):1125-48. doi: 10.1038/nprot.2013.057. Epub 2013 May 16.
This protocol describes the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant antigen-binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.
本方案描述了通过快速鉴定和克隆功能可变重（VH）和可变轻（VL）基因以及设计和克隆针对重组细菌表达进行优化的合成 DNA 序列，从单克隆抗体中衍生出的单价抗原结合分子的设计和开发。通常，单克隆抗体是从小鼠杂交瘤中获得的，这些杂交瘤最常来自免疫小鼠的 B 淋巴细胞与小鼠骨髓瘤细胞的融合。此处描述的方案先前已成功应用于针对广泛生物分子靶标的多种基于抗体的分子的开发。该方案可供可能不专门从事该领域的研究小组使用，并且应该允许在 50 个工作日内从分泌功能性 IgG 的杂交瘤细胞中直接对功能性、重组抗原结合分子进行反向工程。此外，还描述了抗体片段的方便纯化策略。
