Matsuda H, Seo Y, Takahama K
Department of Legal Medicine, Miyazaki Medical College, Japan.
Forensic Sci Int. 1999 Jan 25;99(3):197-208. doi: 10.1016/s0379-0738(98)00194-7.
A sensitive sandwich enzyme immunoassay for human beta-enolase was developed and used to examine beta-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human beta-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human beta-enolase IgG were incubated with human beta-enolase and then with anti-human beta-enolase Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human beta-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of beta-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that beta-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing beta-enolase was recovered well after storage for 60 days at room temperature. The ratio of beta-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of beta-enolase in bloodstains from traumatic blood was obviously elevated (10-30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of beta-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of beta-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that beta-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.
开发了一种用于检测人β-烯醇化酶的灵敏夹心酶免疫分析法，并将其用于检测血液或血迹中的β-烯醇化酶，作为确定骨骼肌损伤的标志物。从人骨骼肌中纯化出人β-烯醇化酶，然后制备针对它的抗体。将包被有兔抗人β-烯醇化酶IgG的聚苯乙烯球与人β-烯醇化酶孵育，然后与抗人β-烯醇化酶Fab'-过氧化物酶缀合物孵育。使用3-(4-羟基苯基)丙酸作为氢供体，通过荧光法测定与聚苯乙烯球结合的过氧化物酶活性。每次测定人β-烯醇化酶的检测限为2.6 pg（30 amol）。除心脏外，该夹心酶免疫分析法对其他器官的交叉反应程度（1/10）约为1/150或更低。此外，通过Northern印迹分析检测了β-烯醇化酶在各种人体组织中的定位，并证实β-烯醇化酶仅在骨骼肌和心肌中表达。含有β-烯醇化酶的血迹在室温下储存60天后，抗原活性恢复良好。由非创伤性血液、鼻出血和月经血制成的血迹中β-烯醇化酶与总蛋白的比值在正常范围内。相比之下，创伤性血液血迹中的β-烯醇化酶比值与非创伤性血液相比明显升高（10-30倍）。此外，事实证明，穿过骨骼肌的武器上附着的血迹中β-烯醇化酶的比值更高，这表明检测血迹中的β-烯醇化酶可用于区分犯罪武器。这些结果表明，β-烯醇化酶是用于识别骨骼肌损伤以及检测出血来源的有用标志物。
