优化酶切抗体片段以提高产量、效率和结合亲和力。
Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.
机构信息
Department of Chemistry , University of Virginia , P.O. Box 400319, Charlottesville , Virginia 22904 , United States.
Department of Biomedical Engineering , University of Virginia , Charlottesville , Virginia 22904 , United States.
出版信息
Bioconjug Chem. 2019 Mar 20;30(3):800-807. doi: 10.1021/acs.bioconjchem.8b00912. Epub 2019 Jan 28.
Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab') production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab') production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.
酶解抗体片段化在各种宿主和同种型中都有很好的研究，但片段化模式也会因克隆而不可预测，通过反复试验来优化 Fab 或 F(ab') 的生产会消耗大量的抗体。在这里，我们报告了一种通过胃蛋白酶消化从小量 IgG 中优化功能性 F(ab') 生产的系统策略。我们测试了三个影响片段化的关键参数，即 pH、酶浓度（%胃蛋白酶 w/w）和反应时间，发现 pH 对片段化产率和效率的影响最大。然后，我们开发了一种系统的方法来获得可接受的产率、消化效率和结合亲和力。描述了三个案例研究来说明该方法。我们预计，这项工作将为研究人员提供一种快速、经济有效的方法，从全 IgG 生产抗体片段，避免随意的反复试验。
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