Gebran S J, Romano E L, Pons H A, Cariani L, Soyano A N
Laboratorio de Fisiopatología, Centro de Medicina Experimental, IVIC, Caracas, Venezuela.
J Immunol Methods. 1992 Jul 6;151(1-2):255-60. doi: 10.1016/0022-1759(92)90125-d.
A sensitive and rapid colorimetric method for the in vitro determination of phagocytic activity and antibody-dependent cell-mediated cytotoxicity (ADCC) is described. The assay uses red blood cells (RBC) as target cells and relies on the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore formed upon oxidation of DAF, was a linear function of erythrocyte concentration. The oxidation of DAF by peritoneal macrophages (M phi) containing myeloperoxidase was negligible, confirming that the development of color was exclusively due to the pseudoperoxidase activity of Hb. A positive correlation was observed between FB formation and increased phagocytosis of opsonized erythrocytes. Phagocytosis increased as a function of time, reaching a maximum at 90 min of incubation. The phagocytosis of IgG-opsonized erythrocytes was greater than non-opsonized erythrocytes and was inhibited by high concentrations of non-specific human or mouse IgG, showing that phagocytosis was mediated by the Fc gamma receptor of macrophages. The interaction between opsonized RBC and macrophages also evoked an antibody-dependent extracellular lysis, however this process was slower than ingestion. The DAF phagocytosis assay has shown to be very sensitive, simple, rapid and safe.
本文描述了一种灵敏、快速的比色法，用于体外测定吞噬活性和抗体依赖性细胞介导的细胞毒性（ADCC）。该检测方法使用红细胞（RBC）作为靶细胞，并依赖于血红蛋白（Hb）的假过氧化物酶活性对2,7-二氨基芴（DAF）进行特异性氧化。DAF氧化生成的发色团芴蓝（FB）是红细胞浓度的线性函数。含有髓过氧化物酶的腹腔巨噬细胞（M phi）对DAF的氧化作用可忽略不计，这证实了颜色的产生完全是由于Hb的假过氧化物酶活性。观察到FB形成与调理素化红细胞吞噬作用增强之间存在正相关。吞噬作用随时间增加，在孵育90分钟时达到最大值。IgG调理素化红细胞的吞噬作用大于未调理素化红细胞，并且被高浓度的非特异性人或小鼠IgG抑制，这表明吞噬作用是由巨噬细胞的Fcγ受体介导的。调理素化红细胞与巨噬细胞之间的相互作用也引发了抗体依赖性细胞外裂解，然而这个过程比摄取要慢。DAF吞噬试验已证明非常灵敏、简单、快速且安全。
