Mohanty J G, Jaffe J S, Schulman E S, Raible D G
MCP Hahnemann School of Medicine, Division of Pulmonary and Critical Care Medicine, Allegheny University of the Health Sciences, Philadelphia, PA 19102, USA.
J Immunol Methods. 1997 Mar 28;202(2):133-41. doi: 10.1016/s0022-1759(96)00244-x.
This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.
本研究描述了一种简单、可靠、高度灵敏且定量的荧光微孔板分析法，用于检测活化白细胞产生的过氧化氢（H2O2），该方法使用了一种新型辣根过氧化物酶（HRP）底物N - 乙酰 - 3,7 - 二羟基吩恶嗪（A6550）。与广泛使用的荧光HRP底物东莨菪亭不同，A6550本身无荧光，在HRP催化H2O2氧化后会产生高强度荧光。使用50微摩尔的A6550时，发现佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯活化的人嗜酸性粒细胞和中性粒细胞产生的H2O2所引起的荧光变化，在细胞剂量分别高达1.5×10⁴和5×10⁴个细胞时呈线性响应。A6550荧光增强是由于H2O2的产生，因为它可被过氧化氢酶抑制。发现氧化后的A6550非常稳定，只要反应混合物中A6550与H2O2的比例高于5，H2O2剂量响应就是线性的。与东莨菪亭不同，A6550背景荧光极低，且随时间变化很小。此外，氧化后的A6550荧光产率高，提高了检测H2O2的灵敏度。当A6550和HRP的浓度分别为10微摩尔和0.2单位/毫升时，低至2皮摩尔的H2O2也能被可靠检测。发现A6550/H2O2分析法的灵敏度比以东莨菪亭作为HRP底物时至少高10倍。本研究中描述的使用A6550测量活化粒细胞释放H2O2的方法，可轻松适用于其他产生H2O2的细胞类型。
