Kraehenbuhl J P, De Grandi P B, Campiche M A
J Cell Biol. 1971 Aug;50(2):432-45. doi: 10.1083/jcb.50.2.432.
The efficiency of small enzyme-labeled tracers for the demonstration of intracellular antigen was investigated in tissues fixed with picric acid-formaldehyde. The influence of fixation on the immunological activity was tested in vitro by radial immunodiffusion. The experimental model consisted of newborn pig jejunum after absorption of ferritin from the intestinal lumen. Ferritin was located after 1 hr in vacuoles scattered in the cytoplasm of the absorptive cells and represented an easily recognizable intracellular antigen. After immunohistochemical treatments with antiferritin preparations, the distribution of labeling enzyme reaction product was examined by morphometry. The ratio of the labeled volume to the total volume of vacuoles containing ferritin indicated the degree of specific labeling of the antigen. In both direct and indirect methods, the degree of labeling was low when enzyme-labeled immunoglobulin G was the tracer. With antigen binding fragments (Fab), the labeling was significantly increased. In the indirect method, the degree of labeling was influenced by the first-step reagents. Onlywhen the serum titer was optimum was a high degree of labeling obtained. With antigen binding fragments or papain-digested serum the effect of the titer was negligible and maximum labeling was achieved. In both methods, with peroxidase as the labeling enzyme, a diffuse nonspecific deposition of reaction product was observed. This could be avoided by using cytochrome c instead.
研究了用苦味酸 - 甲醛固定的组织中，小型酶标记示踪剂用于显示细胞内抗原的效率。通过放射免疫扩散在体外测试固定对免疫活性的影响。实验模型由新生猪空肠组成，该空肠在从肠腔吸收铁蛋白后。1小时后，铁蛋白位于散布在吸收细胞胞质中的液泡中，是一种易于识别的细胞内抗原。在用抗铁蛋白制剂进行免疫组织化学处理后，通过形态计量学检查标记酶反应产物的分布。标记体积与含有铁蛋白的液泡总体积的比率表明了抗原的特异性标记程度。在直接法和间接法中，当酶标记的免疫球蛋白G作为示踪剂时，标记程度较低。使用抗原结合片段（Fab）时，标记显著增加。在间接法中，标记程度受第一步试剂的影响。只有当血清效价最佳时，才能获得高度标记。使用抗原结合片段或木瓜蛋白酶消化的血清时，效价的影响可忽略不计，并可实现最大标记。在两种方法中，当使用过氧化物酶作为标记酶时，观察到反应产物的弥漫性非特异性沉积。改用细胞色素c可避免这种情况。
