Amara Khaled, Israelsson Lena, Stålesen Ragnhild, Sahlström Peter, Steen Johanna, Malmström Vivianne, Grönwall Caroline
Department of Medicine, Rheumatology Division, Karolinska Institutet, and Karolinska University Hospital, Center for Molecular Medicine, Stockholm, Sweden.
Bio Protoc. 2019 Sep 5;9(17):e3347. doi: 10.21769/BioProtoc.3347.
We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both and . This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.
我们在此描述了一种详细、优化的方案，用于从类风湿关节炎（RA）患者的单个B细胞中生成瓜氨酸特异性单克隆抗体。该方案提供了一个详细的操作指南，从您选择的单个B细胞开始，随后通过逆转录聚合酶链反应（RT-PCR）扩增免疫球蛋白基因的可变区，接着进行免疫球蛋白基因克隆、重组IgG1单克隆抗体（mAb）生产及质量控制。所产生的单克隆抗体可用于进一步研究，包括对候选抗原的反应性以及功能研究。该方案可用于从源自不同组织和腔室（包括外周血、滑液、消化活检组织、骨髓穿刺液和支气管肺泡灌洗液）的B细胞中生成抗原特异性单克隆抗体。值得注意的是，尽管给出了如何鉴定瓜氨酸特异性自身抗体的示例，但一般方法也可应用于其他反应性研究。
