Tan W, Yeung E S
Ames Laboratory-U.S. Department of Energy, Iowa 50011.
Anal Biochem. 1995 Mar 20;226(1):74-9. doi: 10.1006/abio.1995.1193.
An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.
已开发出一种基于激光的集成荧光酶测定和颗粒计数免疫测定系统，其具有zeptomole灵敏度，用于同时测定单个红细胞中酶的活性和浓度。监测由葡萄糖-6-磷酸脱氢酶（G6PDH）催化的葡萄糖-6-磷酸与NADP+之间反应的产物NADPH。同时，在G6PDH存在下抗体包被颗粒的凝集产生大颗粒，通过光散射对其进行计数。这两个参数之间的相关性表明，单个细胞中平均只有约35%的酶具有活性。两个参数的比值在细胞间存在超过10倍的差异。这表明作为细胞年龄的函数，细胞内酶的降解存在多种机制。
