U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA.
U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD, USA; Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD, USA; Department of Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.
J Immunol Methods. 2018 Nov;462:74-82. doi: 10.1016/j.jim.2018.08.012. Epub 2018 Aug 24.
Antibody effector functions such as antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) are considered important immunologic parameters following results from the RV144 clinical trial where a reduced risk of infection was associated with non-neutralizing antibody against the V1/V2 region of HIV envelope. The rapid and fluorometric ADCC (RFADCC) assay has been widely used to measure ADCC, however, the mechanism behind the activity measured remains unclear. Here, we demonstrate that monocytes acquire the PKH26 dye used in the RFADCC assay and that the commonly used RFADCC readout correlates with phagocytosis. The RFADCC assay was combined with an amine reactive dye staining to confirm target cell killing. Interestingly, the majority of RFADCC and amine indices were mutually exclusive. In fact, the amine reactive assay results correlated with results from another assays that directly measure NK cell antibody effector functions not associated with phagocytosis. Together, this combined assay offers the opportunity to discriminate monocytes and NK cell antibody effector functions simultaneously.
抗体效应功能，如抗体依赖的细胞细胞毒性（ADCC）和抗体依赖的细胞吞噬作用（ADCP），被认为是重要的免疫学参数，这是基于 RV144 临床试验的结果，该试验表明针对 HIV 包膜的 V1/V2 区域的非中和抗体与感染风险降低有关。快速荧光 ADCC（RFADCC）测定法已被广泛用于测量 ADCC，然而，目前尚不清楚所测量的活性背后的机制。在这里，我们证明单核细胞摄取了 RFADCC 测定法中使用的 PKH26 染料，并且常用的 RFADCC 读数与吞噬作用相关。将 RFADCC 测定法与胺反应性染料染色相结合，以确认靶细胞杀伤。有趣的是，大多数 RFADCC 和胺指数是相互排斥的。实际上，胺反应性测定法的结果与另一种直接测量与吞噬作用无关的 NK 细胞抗体效应功能的测定法的结果相关。总之，这种组合测定法为同时区分单核细胞和 NK 细胞抗体效应功能提供了机会。
