Shin M, Izumi S, Nakane P K
Department of Anatomy III, Nagasaki University School of Medicine, Japan.
J Clin Lab Anal. 1995;9(6):424-30. doi: 10.1002/jcla.1860090615.
A new, extremely sensitive, "multilayer peroxidase-labeled antibody method" is introduced. This method utilizes peroxidase-labeled antibodies that immuno-react with their antigen, but also can be an antigen to other peroxidase-labeled antibodies. An example of this method is: use rabbit IgG as the first antibody, peroxidase-labeled goat antirabbit IgG as the second antibody, peroxidase-labeled rabbit antigoat IgG as the third antibody, peroxidase-labeled goat antirabbit IgG as the fourth antibody, and peroxidase-labeled rabbit antigoat IgG as the fifth antibody in succession. We compared the sensitivities of this multilayer peroxidase-labeled antibody method with that of commercially available immuno-peroxidase staining kits, i.e., avidin-biotin-complex method and peroxidase-antiperoxidase method, both by immunostaining and enzyme-linked immunosorbent assay. The generated signal and background in immunostained sections were determined by a computer-assisted image analyzer and by enzyme-linked immunosorbent assay spectrophotometrically. It was found that a maximum signal/background ratio was obtained with those sections reacted with the first four antibodies in succession by the multilayer peroxidase-labeled antibody method, and those reacted with the five antibodies in succession by the multilayer peroxidase-labeled antibody method in the enzyme-linked immunosorbent assay. Thus, the multilayer peroxidase-labeled antibody method is more sensitive than the avidin-biotin-complex method and the peroxidase-antiperoxidase method.
一种新的、极其灵敏的“多层过氧化物酶标记抗体法”被引入。该方法利用与抗原发生免疫反应的过氧化物酶标记抗体，同时该抗体也可作为其他过氧化物酶标记抗体的抗原。此方法的一个实例是：依次使用兔IgG作为一抗，过氧化物酶标记的山羊抗兔IgG作为二抗，过氧化物酶标记的兔抗山羊IgG作为三抗，过氧化物酶标记的山羊抗兔IgG作为四抗，以及过氧化物酶标记的兔抗山羊IgG作为五抗。我们通过免疫染色和酶联免疫吸附测定法，将这种多层过氧化物酶标记抗体法的灵敏度与市售免疫过氧化物酶染色试剂盒（即抗生物素蛋白-生物素复合物法和过氧化物酶-抗过氧化物酶法）的灵敏度进行了比较。免疫染色切片中产生的信号和背景通过计算机辅助图像分析仪测定，并通过酶联免疫吸附测定法进行分光光度测定。结果发现，在多层过氧化物酶标记抗体法中，依次与前四种抗体反应的切片以及在酶联免疫吸附测定中依次与五种抗体反应的切片获得了最大的信号/背景比。因此，多层过氧化物酶标记抗体法比抗生物素蛋白-生物素复合物法和过氧化物酶-抗过氧化物酶法更灵敏。
