Mompó Susana Magadán, González-Fernández Africa
Immunology, Biomedical Research Center (CINBIO), Vigo, Spain.
Methods Mol Biol. 2014;1060:245-76. doi: 10.1007/978-1-62703-586-6_13.
Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germ line configuration. The engineered mouse genome can undergo productive rearrangement in the B cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animals expressing human Ig genes. This chapter describes the type of transgenic knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.
由于通过传统方法生成完全人源单克隆抗体（mAb）时发现存在困难，人们进行了多项努力试图克服这些问题，取得了不同程度的成功。一种方法是开发在种系构型中携带免疫球蛋白（Ig）基因的转基因小鼠。经过基因工程改造的小鼠基因组能够在B细胞群体中进行有效的重排，产生表达人Ig（hIg）链的小鼠B淋巴细胞。为避免小鼠重链或轻链的表达，必须通过基因靶向技术使内源性小鼠Ig（mIg）基因座沉默。随后，为获得抗原特异性单克隆抗体，可以遵循常规免疫方案，并对这些表达人Ig基因的动物使用单克隆抗体技术（将活化的B细胞与小鼠骨髓瘤细胞融合、筛选、克隆、冷冻和测试）。本章描述了为各个研究小组生成的转基因敲除小鼠的类型，提供了研究小组和公司开发的人源单克隆抗体的实例，并包括从这类小鼠免疫、生成、生产和纯化人源单克隆抗体的方案。此外，它还讨论了检测到的问题，并包括一些可用于分析人源单克隆抗体功能活性的方法。
