Zelechowska M G, Mandeville R
Immunology Research Center, Institut Armand-Frappier, Laval-des-Rapides, Quebec, Canada.
Anticancer Res. 1989 Jan-Feb;9(1):53-7.
A new method for demonstrating the binding and internalization of target molecules identified by two different monoclonal antibodies (MAbs) is described. This double staining technique utilizes, in a pre-embedding procedure, an immunogold/silver staining and a MAb that recognized cell surface antigens and a post-embedding technique where only immunogold is used to identify intracytoplasmic antigens. We could demonstrate that immunogold and gold followed by silver enhancement are two highly sensitive and accurate techniques. Colloidal gold particles are versatile tracers at the electron microscopic level when used at two different particle sizes (5 and 20 nm in diameter) in a double labelling method for the simultaneous identification of antigenic sites on the same section; the combined use of immunogold and gold/silver staining for the simultaneous localization of breast cancer-associated antigens on the same thin section is more accurate. These techniques have also been used to visualize the internationalization of antigen-antibody complexes by incubating MAb-gold labelled tissue sections for 30 min at 37 degrees C. We conclude that the use of both colloidal gold and gold/silver constitutes a further improvement in immunoelectron microscopy techniques and can help visualize the relative pattern of reactivity of two MAbs on the same cells. Further applications of these techniques will have an important impact on the development and use of MAbs in oncology.
本文描述了一种用于展示由两种不同单克隆抗体（MAb）识别的靶分子的结合和内化的新方法。这种双重染色技术在包埋前程序中利用免疫金/银染色和一种识别细胞表面抗原的单克隆抗体，在包埋后技术中仅使用免疫金来识别胞浆内抗原。我们能够证明免疫金和随后进行银增强的金是两种高度灵敏且准确的技术。当在双重标记方法中使用两种不同粒径（直径分别为5和20纳米）的胶体金颗粒在同一切片上同时识别抗原位点时，胶体金颗粒在电子显微镜水平上是通用的示踪剂；在同一薄切片上联合使用免疫金和金/银染色同时定位乳腺癌相关抗原更为准确。这些技术还通过在37℃下将单克隆抗体-金标记的组织切片孵育30分钟来观察抗原-抗体复合物的内化情况。我们得出结论，胶体金和金/银的使用构成了免疫电子显微镜技术的进一步改进，并且有助于观察两种单克隆抗体在同一细胞上的相对反应模式。这些技术的进一步应用将对肿瘤学中单克隆抗体的开发和使用产生重要影响。
