Sumiko Abe

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

Abstract A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-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. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10 7 times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-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 γ-GTP was detectable even in 14-year-old stains.

Purification and immunological characterization of a new form of γ-glutamyltransferase of human semen

A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal γ-glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal γ-glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal γ-glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal γ-glutamyltransferase but recognize different epitopes.

A sandwich enzyme-linked immunosorbent assay for human seminal γ-glutamyl transpeptidase

Abstract A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting human seminal γ-glutamyl transpeptidase (γ-GTP) using a combination of anti-seminal γ-GTP monoclonal antibodies. These monoclonal antibodies did not react with human ovary or uterus in immunohistochemical study. Optimal assay condition, resulting in a sensitive assay with a low background, is presented. The detection limit of this assay was estimated to be 1 ng/ml of seminal γ-GTP corresponding to about 100 000 times dilution of seminal sample. This ELISA was specific for seminal γ-GTP, without cross-reactivity to renal or hepatic gg-GTP, normal blood serum, non-coital vaginal fluid or saliva. The recovery of seminal γ-GTP added to various biological fluids were also examined.

Polymorphism of seminal γ-glutamyl transpeptidase

Abstract Electrophoretic analysis of seminal γ-glutamyl transpeptidase (GGT) activity of 147 unrelated Japanese males revealed three types of band patterns. An anodal single band, a cathodal single band and heterozygous double bands termed 1, 2 and 2-1, respectively, were commonly identified in the samples. The frequencies of the three types were 1 = 0.22, 2 = 0.33 and 2-1 = 0.44. Seminal stains kept for more than 6 months revealed distinguishable band patterns as well as fresh samples.

Dimethylthiourea ameliorates carbon tetrachloride-induced acute liver injury in ovariectomized mice

AIMS In order to clarify hepato-protective actions of estrogen, we examined the progress of carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in sham and ovariectomized (ovx) mice and the effects of dimethylthiourea (DMTU), a hydroxyl radical scavenger, and meloxicam (Melo), a selective cox-2 inhibitor, on the development of CCl4-induced ALI. MAIN METHODS Female C57BL/6 J mice weighing 15-20 g were performed sham or ovx operation at 8 weeks of age. Blood and liver samples were collected 15 and 24 h after CCl4 administration. Sham and ovx mice were given DMTU, Melo or saline intraperitoneally 30 min before CCl4 or corn oil administration. KEY FINDINGS ALT levels in ovx mice were significantly increased compared to those in sham mice. DMTU reduced ALT levels in ovx mice to the same levels as those in sham mice after CCl4 injection. CCl4 upregulated TNF-α, IL-6, cox-2 and iNOS expression in ovx mice compared to the levels in sham mice. DMTU significantly reduced cox-2 and iNOS expression levels upregulated by CCl4 in ovx mice. However, pretreatment with Melo had no effects on ALT levels and the gene expression levels of TNF-α, IL-6 and HO-1 in either sham or ovx mice, indicating that cox-2 may not participate in increase of CCl4-induced ALI caused by estrogen deficiency. SIGNIFICANCE Ovariectomy accelerated the development of CCl4-induced acute liver injury, and DMTU reduced liver injury. These results suggest that estrogen may act as an antioxidant in the development CCl4-induced acute liver injury.

Alpha-2-HS-glycoprotein phenotype frequencies in Cook Islanders.

The polymorphism of the alpha 2-HS-glycoprotein (A2HS) was analysed in Rarotonga and Mangaia, the Cook Islands. The A2HS*2 frequency was found to be the highest value among all populations studied up to now. There was a significant difference in A2HS*2 gene frequencies between the two populations, Rarotonga (0.62) and Mangaia (0.76).