Díez Paula, Jara-Acevedo Ricardo, González-González María, Casado-Vela Juan, Dasilva Noelia, Lécrevisse Quentin, Bartolomé Raquel, Claros José Carlos, González Alfredo, López Ricardo, Orfao Alberto, Fuentes Manuel
Cancer Research Center, University of Salamanca-CSIC, IBSAL, Department of Medicine, General Service of Cytometry, Campus Miguel de Unamuno S/N, 37007 Salamanca, Spain; Proteomics Unit, Cancer Research Center, IBSAL, University of Salamanca-CSIC, Campus Miguel de Unamuno s/n, 37007 Salamanca, Spain.
ImmunoStep SL, Cancer Research Center, Avda, Universidad de Coimbra S/N, 37007 Salamanca, Spain.
Enzyme Microb Technol. 2015 Nov;79-80:34-41. doi: 10.1016/j.enzmictec.2015.06.016. Epub 2015 Jul 14.
Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.
用于设计和生成人源抗体的新兴技术需要改进方法，以实现对其进行筛选、表征和验证。目前，基于酶联免疫吸附测定（ELISA）或蛋白质免疫印迹法（western blot）的策略被用于该目的。然而，不断增加的新型抗体的产生将受益于新的高通量（HT）平台的开发，这些平台有助于快速鉴定和表征抗体。在此，我们描述了一种承载重组噬菌体颗粒并基于大型噬菌体抗体库的蛋白质芯片。在本文中，我们提出了一种新颖的实施方案，该方案提供了一种强大而简单的方法，能够鉴定单链可变片段（scFv）。作为该方法的原理验证，我们用重组抗原（人重组白细胞介素8）对其进行了测试。此外，我们开发了一种新颖的生物信息学工具，用于将这种新策略与传统方法进行比较。本文所述的方法以及相关的信息学工具是可靠的、相对快速的，并且代表了包括噬菌体库筛选在内的实验方案向前迈进的一步。
