Corsiero Elisa, Jagemann Lucas, Bombardieri Michele, Pitzalis Costantino
Centre for Experimental Medicine and Rheumatology, John Vane Science Centre, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
Methods Mol Biol. 2018;1845:159-187. doi: 10.1007/978-1-4939-8709-2_10.
Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.
异位淋巴结构（ELS）可在类风湿关节炎（RA）和干燥综合征（SS）等慢性炎症性自身免疫病患者的靶组织中形成。尽管目前尚不清楚为何ELS仅在部分患者中形成，但众所周知，这些结构可获得异位生发中心的特征，并积极参与自身抗体的产生。在此，我们描述了一种从RA患者的单个ELS滑膜组织B细胞中产生重组单克隆抗体的方法。本章详细介绍了该方法，从RA滑膜组织的单核细胞制备开始，通过流式细胞术对B细胞进行单细胞分选，通过聚合酶链反应（PCR）扩增免疫球蛋白（Ig）基因（重链和轻链基因），随后进行Ig基因表达载体克隆，以在体外产生完全重组的IgG1单克隆抗体（rmAb）。然后可对产生的重组单克隆抗体进行如下表征：（1）用于克隆研究的Ig基因库分析；（2）免疫反应性谱分析；（3）体内外功能研究。
