The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method
The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method
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DOI：
10.4161/mabs.27044
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发表时间：
2014-01-01
期刊：
影响因子：
5.3
通讯作者：
Lightwood, Daniel J.
中科院分区：
文献类型：
--
作者：
Clargo, Alison M.;Hudson, Ashley R.;Lightwood, Daniel J.
单B细胞技术，避免了传统的杂交瘤融合和组合展示，提供了一种方法来询问免疫动物的天然选择的抗体库。许多方法能够对记忆B细胞亚群进行取样，但很少允许直接询问浆细胞库，即，负责在血清中产生免疫球蛋白的B细胞亚群。在这里，我们描述了使用一个强大的和简单的荧光为基础的技术，称为荧光灶的方法，用于识别和分离抗原特异性IgG分泌细胞，如浆细胞，从异质性骨髓制剂。显微操作单细胞后，通过逆转录（RT）-聚合酶链反应（PCR）回收重链和轻链可变区基因的同源对。在PCR期间，将可变区与启动子片段和相关恒定区片段组合以产生两个单独的转录活性PCR（TAP）片段，将其直接共转染到HEK-293 F细胞系中用于重组抗体表达。该技术被成功地应用于生成一个不同的面板的高亲和力，功能性重组抗体的人肿瘤坏死因子（TNF）受体2和TNF来自免疫兔和大鼠的骨髓，分别。从骨髓样本到一组功能性重组抗体的进展可能在2周时间范围内完成。
Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.