A novel approach for rapid high-throughput selection of recombinant functional rat monoclonal antibodies.
A novel approach for rapid high-throughput selection of recombinant functional rat monoclonal antibodies.
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一种快速高通量选择重组功能性大鼠单克隆抗体的新方法
DOI：
10.1186/s12865-018-0274-8
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发表时间：
2018-12-04
期刊：
影响因子：
3
通讯作者：
Huang C
中科院分区：
文献类型：
--
作者：
Chen Q;Qiu S;Li H;Lin C;Luo Y;Ren W;Zou Y;Wang Y;Xia N;Huang C
背景大多数抗小鼠抗原的单克隆抗体来源于大鼠脾-小鼠骨髓瘤融合体，这是从一般实验室试剂到治疗药物的有价值的工具，然而选择和表达它们仍然是一个耗时且低效的过程。在这里，我们报告了一种新的方法，用于快速高通量选择和表达的重组功能大鼠单克隆抗体与不同的isotype.ResultsWe已经开发出一个强大的系统，用于产生大鼠单克隆抗体，通过几个过程，涉及同时免疫大鼠与三种不同的抗原表达在混合细胞池，制备杂交瘤细胞池，使用高效组装方法体外筛选并随后将重排的轻链和重链克隆到单个表达质粒中，这可以减少多次免疫和融合、传统克隆选择和表达方法所需的时间和精力。使用该系统，我们成功地从单个融合物中选择了几种具有不同IgG同种型的大鼠单克隆抗体，其特异性靶向小鼠PD-1、LAG-3或AFP蛋白。我们应用这些重组抗PD-1单克隆抗体（32 D 6）在免疫治疗的治疗目的，产生了预期的results.ConclusionsThis方法可用于促进增加吞吐量的整个过程中，从多重免疫功能的大鼠单克隆抗体的收购，并促进其表达和可行性，使用一个单一的质粒。
BackgroundMost monoclonal antibodies against mouse antigens have been derived from rat spleen-mouse myeloma fusions, which are valuable tools for purposes ranging from general laboratory reagents to therapeutic drugs, and yet selecting and expressing them remains a time-consuming and inefficient process. Here, we report a novel approach for the rapid high-throughput selection and expression of recombinant functional rat monoclonal antibodies with different isotypes.ResultsWe have developed a robust system for generating rat monoclonal antibodies through several processes involving simultaneously immunizing rats with three different antigens expressing in a mixed cell pools, preparing hybridoma cell pools, in vitro screening and subsequent cloning of the rearranged light and heavy chains into a single expression plasmid using a highly efficient assembly method, which can decrease the time and effort required by multiple immunizations and fusions, traditional clonal selection and expression methods. Using this system, we successfully selected several rat monoclonal antibodies with different IgG isotypes specifically targeting the mouse PD-1, LAG-3 or AFP protein from a single fusion. We applied these recombinant anti-PD-1 monoclonal antibodies (32D6) in immunotherapy for therapeutic purposes that produced the expected results.ConclusionsThis method can be used to facilitate an increased throughput of the entire process from multiplex immunization to acquisition of functional rat monoclonal antibodies and facilitate their expression and feasibility using a single plasmid.
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