Department of Immunology, Genetics and Pathology, Science for Life Laboratory, BMC, Uppsala University, Uppsala, Sweden.
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Mod Pathol. 2018 Feb;31(2):253-263. doi: 10.1038/modpathol.2017.102. Epub 2017 Sep 22.
Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.
抗体是解剖病理学和研究中的重要工具，但免疫组织化学中对原位蛋白质的检测质量在很大程度上取决于抗体的选择和目标蛋白质的丰度。许多用于科学研究的抗体不符合特异性和灵敏度的要求。因此，改进抗体性能并产生定量数据的方法可以极大地促进基于蛋白质表达和原位定位的科学研究和临床诊断。我们在这里展示了抗体标记的方案，允许通过明场原位邻近连接测定法在组织中特异性检测蛋白质，其中每个蛋白质分子必须被两种抗体识别。我们进一步证明，这些双识别测定可以使用单克隆抗体或纯化的血清制剂。抗体缀合物对蛋白质的成对识别要求可以显著提高蛋白质检测的特异性，超过单结合物测定。
