酶标荧光-97检测中性粒细胞碱性磷酸酶活性的新方法

ENZYME LABELED FLUORESCENCE 97 TO DETECT THE ACTIVITY OF ALKALINE PHOSPHATASE IN NEUTROPHILS

目的用一种新试剂酶标荧光-97(ELF-97)检测人血中性粒细胞碱性磷酸酶(NAP)活性,观察NAP阳性细胞数及阳性颗粒形态和分布。方法用Ficoll为介质的等密度沉降分离法从全血中分离出中性粒细胞,固定后以ELF-97为底物进行酶细胞化学反应,荧光/微分干涉相差显微镜观察并摄片。结果荧光显微镜下可见中性粒细胞内有明亮的黄绿色荧光颗粒,颗粒在细胞内的分布均匀,大小不一,51例健康成人血样NAP阳性率为(94.102±3.133)%。结论ELF-97荧光检测法操作简便,灵敏度高,产物不容易扩散,重复性好,结果精确,可作为不同用途的碱性磷酸酶检测手段。

Objective To localize endogenous alkaline phesphatase in human neutrophils with ELF （enzyme-labeled fluorescence）-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. Methods Neutrophils were isolated from human blood and fixed, then histochemistry with the ELF-97 phosphate was performed with the ELF-97 endogenous phosphatase detection kit. All photography was performed with a Nikon labophot fluorescence/DIC microscope. Results Fluorescent yellow-green granules, well-distributed but size-varied, were observed in neutrophils under fluorescence microscope.There were 94. 102± 3.133 percent rate of positive neutrophil alkaline phosphatase among 51 cases of healthy volunteers. Conclusion ELF-97 endogenous phosphatase detection kit can provide sensitive, high-resolution localization of endogenous phosphatase activity in neutrophils.