Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.
Reliable and controllable antibody fragment selections from Camelid non-immune libraries for target validation.
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从 Camelid 非免疫文库中选择可靠且可控的抗体片段，用于靶标验证。
DOI：
10.1016/j.bbapap.2006.05.011
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
vanderMaarel,SilvèreM
中科院分区：
文献类型：
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作者：
Verheesen,Peter;Roussis,Andreas;deHaard,HansJ;Groot,ArjanJ;Stam,JordC;denDunnen,JohanT;Frants,RuneR;Verkleij,ArieJ;TheoVerrips,C;vanderMaarel,SilvèreM
随着人类基因组序列的完成，现在的重点转向了人类蛋白质组。然而，转录组中蛋白质的数量不仅大于mrna，而且蛋白质通常需要与其他蛋白质复合物才能发挥功能。在自然环境下研究蛋白质的一个有利选择是结合使用特异性抗体的生化和显微技术。为此，我们设计了一种快速、可靠、可控的从骆驼非免疫文库中筛选单域抗体片段（VHH）的方法。我们分离了4种肌肉疾病相关蛋白的VHH；emerin, actin，原肌球蛋白-1和核聚(A)结合蛋白。抗体对靶标验证研究的重要特征是识别抗原的自然构象和生物相关的复合物。我们表明所选择的抗体片段在各种免疫学技术中具有功能，并证明在诊断应用中是有用的。我们的选择策略适用于自动化和蛋白质组学平台的建立。它为快速和经济有效地获得许多抗原的多个抗体片段开辟了道路，这些抗原可以检测其定位，水平和修饰的变化以及通常与疾病相关的超分子结构的细微变化。
With the completion of the sequence of the human genome, emphasis is now switching to the human proteome. However, the number of proteins is not only larger than mRNAs in the transcriptome, proteins need often to be in complex with other proteins to be functional. A favourable option to study proteins in their natural context is with a combination of biochemical and microscopic techniques using specific antibodies. Therefore, we designed a fast, reliable and controllable selection and screening of single-domain antibody fragments (VHH) from a Camelid non-immune library. We isolated VHH for four muscle disease related proteins; emerin, actin, tropomyosin-1, and nuclear poly(A)-binding protein. Important features of antibodies for target validation studies are recognition of the antigen in natural conformations and biologically relevant complexes. We show that selected antibody fragments are functional in various immunological techniques and prove useful in diagnostic applications. Our selection strategy is amenable to automation and to the establishment of proteomics platforms. It opens the way to quickly and cost-effectively obtain multiple antibody fragments for many antigens that can detect changes in their localization, level, and modification as well as subtle changes in supramolecular structures, which often associate with disease.