Hamilton S, Healy M, Corris P, Scott R
Department of Virology, University of Newcastle Upon Tyne, Medical School, UK.
Histochem J. 1995 Jun;27(6):487-93.
An immunohistochemical method for assessing the level of tumour necrosis factor-alpha in alveolar macrophages obtained by brochoalveolar lavage is described. Cytospins of mixed populations of lung cells were incubated first with a monoclonal antibody to CD68 and then with a specific peroxidase-labelled second antibody in a two-step reaction for the detection of the macrophage marker CD68. A second similarly based two-step reaction for the detection of tumour necrosis factor-alpha followed. Both reactions were visualized, on completion, using different coloured peroxidase substrates which produced a third colour in the event of dual deposition of the substrates. Dual substrate deposition was indicative of alveolar macrophages positive for tumour necrosis factor-alpha. This method has provided a specific and reproducible semi-quantitative test for the presence of tumour necrosis factor-alpha in human activated alveolar macrophages, which can be performed retrospectively on clinical material. A range of concentrations of the cytokine has been demonstrated in individual samples. This dual detection method has the potential for detection of any cell-associated protein product by minor modification of the described method.
本文描述了一种免疫组织化学方法，用于评估通过支气管肺泡灌洗获得的肺泡巨噬细胞中肿瘤坏死因子-α的水平。首先将混合的肺细胞群体的细胞涂片与抗CD68单克隆抗体孵育，然后在两步反应中与特异性过氧化物酶标记的二抗孵育，以检测巨噬细胞标志物CD68。随后进行基于类似原理的另一个两步反应，用于检测肿瘤坏死因子-α。完成后，使用不同颜色的过氧化物酶底物使两个反应显色，如果底物双重沉积则会产生第三种颜色。双重底物沉积表明肿瘤坏死因子-α呈阳性的肺泡巨噬细胞。该方法为检测人活化肺泡巨噬细胞中肿瘤坏死因子-α的存在提供了一种特异性且可重复的半定量检测方法，可对临床材料进行回顾性检测。已在单个样本中证实了一系列细胞因子浓度。通过对所述方法进行微小修改，这种双重检测方法有可能检测任何细胞相关蛋白产物。
