Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA; Oak Ridge Institute for Science and Education (ORISE), CDC Fellowship Program, Oak Ridge, TN, USA.
Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA.
J Immunol Methods. 2024 Nov;534:113768. doi: 10.1016/j.jim.2024.113768. Epub 2024 Oct 22.
Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available in silico. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 μg (14-98 μg) after 5 days in 2 ml cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.
单克隆抗体是强大的治疗、诊断和研究工具。生成单克隆抗体的方法正在迅速发展。我们从合成 DNA 片段的 2 小时高保真重叠 PCR 反应中创建了一个可转染的线性抗体表达盒。我们将重链和轻链与启动子、自我切割肽和 poly(A)信号连接成一个单一的线性序列，以增加从任何可用的序列中交换可变区的灵活性。线性盒的转染往往会产生与两质粒系统相似的水平，并在 2 毫升培养物中培养 5 天后产生平均 47μg（14-98μg），有 15 个独特的抗体序列。在不到一周的时间内，产生的抗体水平足以满足大多数下游应用。这里介绍的方法减少了克隆步骤的时间、成本和复杂性。
