Van der Loos C M, Das P K, Van den Oord J J, Houthoff H J
Department of Pathology, Academic Medical Centre, Amsterdam, The Netherlands.
J Immunol Methods. 1989 Feb 8;117(1):45-52. doi: 10.1016/0022-1759(89)90117-8.
Simultaneous detection of multiple tissue epitopes with an overlapping distribution pattern by monoclonal antibodies is sometimes needed for routine immunohistological evaluations. Therefore, multistep double and triple immunoenzymatic methods using antibodies from the same species or Ig (sub)class have been developed. Since only commercially available monoclonal antibodies (either unlabelled, biotinylated or as fluorescein conjugate) have been used, the techniques may be regarded as generally applicable. The staining protocol for double staining consists of six incubation steps: (1) unlabelled monoclonal antibody 1; (2) enzyme I-conjugated anti-mouse Ig; (3) normal mouse serum--for blocking; (4) fluoresceinated monoclonal antibody 2; (5) rabbit anti-fluorescein isothiocyanate--employing the fluorochrome as hapten; (6) enzyme II-conjugated anti-rabbit Ig. For enzymes I and II, peroxidase, alkaline phosphatase and beta-galactosidase can be applied; excellent results were obtained with the following colour combinations: peroxidase activity in red/alkaline phosphatase in blue and beta-galactosidase in green/alkaline phosphatase in violet. Moreover, this double staining method can be extended to provide an immunoenzyme triple staining technique by mixing biotinylated monoclonal antibody 3 and avidin-biotin enzyme III complex with the steps 4 and 5 reagents, respectively. In this way three tissue epitopes can simultaneously be detected clearly and selectively in green (beta-galactosidase), blue (alkaline phosphatase) and red (peroxidase).
在常规免疫组织学评估中，有时需要用单克隆抗体同时检测具有重叠分布模式的多种组织表位。因此，已开发出使用来自同一物种或Ig（亚）类的抗体的多步双重和三重免疫酶法。由于仅使用了市售单克隆抗体（未标记、生物素化或荧光素缀合物形式），这些技术可被视为普遍适用。双重染色的染色方案包括六个孵育步骤：（1）未标记的单克隆抗体1；（2）酶I缀合的抗小鼠Ig；（3）正常小鼠血清——用于封闭；（4）荧光素化的单克隆抗体2；（5）兔抗异硫氰酸荧光素——将荧光染料用作半抗原；（6）酶II缀合的抗兔Ig。对于酶I和酶II，可应用过氧化物酶、碱性磷酸酶和β-半乳糖苷酶；通过以下颜色组合获得了优异结果：红色中的过氧化物酶活性/蓝色中的碱性磷酸酶活性以及绿色中的β-半乳糖苷酶活性/紫色中的碱性磷酸酶活性。此外，通过分别将生物素化的单克隆抗体3和抗生物素蛋白-生物素酶III复合物与步骤4和5的试剂混合，这种双重染色方法可扩展为提供一种免疫酶三重染色技术。通过这种方式，可以在绿色（β-半乳糖苷酶）、蓝色（碱性磷酸酶）和红色（过氧化物酶）中同时清晰且选择性地检测到三种组织表位。
