Signore Michele, Manganelli Valeria, Hodge Alex
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Via Giano della Bella 34, 00162, Rome, Italy.
Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.
Methods Mol Biol. 2017;1606:51-70. doi: 10.1007/978-1-4939-6990-6_4.
Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).
抗体验证是转化研究不可或缺的一部分，尤其是在生物标志物发现方面。在临床研究中应用任何生物标志物之前，检测试剂（抗体）的特异性并确认蛋白质生物标志物的身份至关重要，缺乏此类质量控制测试可能会导致意外和/或误导性结果。抗体验证是对单一抗体的敏感性和特异性进行全面检测的过程。尽管有大量商业抗体存在，但在临床转化研究中使用之前，必须使用多种复杂生物样品而非纯化的重组蛋白来广泛证明抗体的特异性。在最简单的情况下，抗体特异性通过在复杂生物样品中、预期分子量处的蛋白质印迹上出现单一条带来确定。
迄今为止，基于手动或自动化系统，有许多蛋白质印迹方法可供使用，涵盖从单印迹到多重印迹的范围。许多研究实验室仍在使用X射线胶片，但数字成像已成为免疫印迹的金标准。蛋白质印迹的基本原理是：（a）通过凝胶电泳分离蛋白质混合物；（b）将蛋白质转移到印迹膜上；（c）用针对感兴趣的一种或多种蛋白质的探针检测印迹膜；（d）随后通过化学发光、荧光或比色法检测蛋白质。本章重点介绍使用手动蛋白质印迹系统和真空增强检测系统（SNAP i.d.™，密理博公司）对蛋白质进行化学发光检测。
