Background: Most monoclonal antibodies against mouse antigens have been derived from rat spleen-mouse\nmyeloma fusions, which are valuable tools for purposes ranging from general laboratory reagents to therapeutic\ndrugs, and yet selecting and expressing them remains a time-consuming and inefficient process. Here, we report a\nnovel approach for the rapid high-throughput selection and expression of recombinant functional rat monoclonal\nantibodies with different isotypes.\nResults: We have developed a robust system for generating rat monoclonal antibodies through several processes\ninvolving simultaneously immunizing rats with three different antigens expressing in a mixed cell pools, preparing\nhybridoma cell pools, in vitro screening and subsequent cloning of the rearranged light and heavy chains into a\nsingle expression plasmid using a highly efficient assembly method, which can decrease the time and effort\nrequired by multiple immunizations and fusions, traditional clonal selection and expression methods. Using this\nsystem, we successfully selected several rat monoclonal antibodies with different IgG isotypes specifically targeting\nthe mouse PD-1, LAG-3 or AFP protein from a single fusion. We applied these recombinant anti-PD-1 monoclonal\nantibodies (32D6) in immunotherapy for therapeutic purposes that produced the expected results.\nConclusions: This method can be used to facilitate an increased throughput of the entire process from multiplex\nimmunization to acquisition of functional rat monoclonal antibodies and facilitate their expression and feasibility\nusing a single plasmid.
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