Detection of human seminal gamma-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal gamma-glutamyl transpeptidase (gamma-GTP) was developed using a combination of monoclonal antibodies. SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. gamma-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal gamma-GTP equivalent to 10(7) times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal gamma-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal gamma-GTP was detectable even in 14-year-old stains.