A sandwich enzyme immunoassay for human muscle-specific beta-enolase and its application for the determination of skeletal muscle injury.
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.
Matsuda
H
H
Department of Legal Medicine, Miyazaki Medical College, Japan.
Seo
Y
Y
Takahama
K
K
D000818:Animals; D001797:Blood Protein Electrophoresis; D001801:Blood Stains; D015152:Blotting, Northern; D003429:Cross Reactions; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000293:injuries*; D010751:Phosphopyruvate Hydratase / Q000032:analysis*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D010751:Phosphopyruvate Hydratase