Birgersson Madeleine, Katona Borbala, Lindskog Cecilia, Pontén Fredrik, Williams Cecilia
Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Methods Mol Biol. 2022;2418:1-23. doi: 10.1007/978-1-0716-1920-9_1.
Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.
抗体可能会与预期靶点以外的蛋白质发生交叉反应，如果基于抗体的应用未得到适当验证，可能会导致有缺陷的解释。2017年在评估13种抗雌激素受体β（ERβ）抗体时，我们得出结论，其中只有一种具有特异性。将这种抗体应用于44种以上不同的正常人体组织和20种癌症的免疫组织化学分析中，结果显示仅在少数特定组织中存在ERβ表达。这与mRNA证据相符，但与大量已发表的文献相矛盾。在没有mRNA表达明确支持的情况下，仍不断有文献报道某些组织中存在ERβ蛋白表达。在本章中，我们描述了如何对ERβ抗体进行全面验证，并讨论了如何选择特征明确的阳性和阴性对照。所介绍的验证方案适用于免疫组织化学和蛋白质印迹法。该方案包括评估mRNA证据、使用公共数据库、评估靶向和非靶向结合，以及一个用于通过免疫沉淀和质谱法进行确证的可选步骤。
