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Serum des-gamma-carboxy prothrombin (DCP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), but the exact mechanism of its synthesis and its structural properties in liver diseases are unknown. DCP is measured by the monoclonal antibody MU-3. The purpose of this study was to examine the epitope of MU-3 and to characterize the differences in DCP between HCC and benign liver diseases. The epitope of MU-3 was examined by ELISA using prothrombin Gla domain polypeptides and was determined to be amino acid residues 17-27 of the prothrombin Gla domain, which has four gamma-carboxyglutamic acid residues (Gla) at positions 19, 20, 25 and 26. Peptides having a glutamic acid residue (Glu) at these positions reacted strongly to MU-3 but lost reactivity when Glu 19 or 20 was changed to Gla. In the order of gamma-carboxylation, MU-3 reacted strongly to DCP containing 0-1 Gla, weakly to 2-4 Gla and not at all to DCP containing more than five Gla. After adsorbing normal prothrombin with barium carbonate, DCP reaction to MU-3 was measured by determining the amount of DCP that was adsorbed by MU-3-coated beads. The proportion of DCP reacting to MU-3 in HCC was 41.0-76.8%, whereas in patients with benign liver diseases, only 0-42.1% reacted to MU-3. These results indicate that DCP variants preferentially synthesized in HCC have less than four Gla, which are restricted to positions 16, 25, 26 and 29, whereas DCP variants in benign liver diseases have more than five Gla.

The purpose of this study is to determine serum des-gamma-carboxy prothrombin (DCP) levels in benign liver diseases by a new sensitive method, and to demonstrate the elevation of serum DCP in alcoholic liver disease (ALD) without hepatocellular carcinoma (HCC). Median values of serum DCP were 16.2 mAU/ml (range: 3.2 to 1570 mAU/ml) in ALD and 16.7 mAU/ml (1.2 to 75.4 mAU/ml) in viral liver disease (VLD). Using the cut-off value of 40 mAU/ml as a tumor marker for HCC, 21% (11/52) was positive in ALD and 2% (1/57) was positive in VLD (p = 0.0014, Fisher's exact probability test), and 27% (9/33) was positive in alcoholic liver cirrhosis and 3% (1/39) was positive in viral liver cirrhosis (p = 0.0042, Fisher's exact probability test). The positive rate of DCP was significantly (p < 0.001, Spearman's rank correlation test) correlated with the severity of liver disease in ALD. Serum vitamin K level was not decreased in cases with ALD. In a demonstrable case, serum DCP was decreased after abstinence and was increased again after the beginning of ethanol intake, suggesting the involvement of ethanol to the elevation of serum DCP in ALD. In conclusion, serum DCP was significantly elevated in ALD, compared with VLD, although the mechanism of the elevation of DCP was not clarified. Ethanol intake may act, in part, on the increase of serum DCP in ALD.

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.

The prevalence of Borna disease virus (BDV)-specific antibodies among patients with psychiatric disorders and healthy individuals has varied in several reports using several different serological assay methods. A reliable and specific method for anti-BDV antibodies needs to be developed to clarify the pathological significance of BDV infections in humans. We developed a new electrochemiluminescence immunoassay (ECLIA) for the antibody to BDV that uses two recombinant proteins of BDV, p40 and p24 (full length). Using this ECLIA, we examined 3,476 serum samples from humans with various diseases and 917 sera from blood donors in Japan for the presence of anti-BDV antibodies. By ECLIA, 26 (3.08%) of 845 schizophrenia patients and 9 (3.59%) of 251 patients with mood disorders were seropositive for BDV. Among 323 patients with other psychiatric diseases, 114 with neurological diseases, 75 with chronic fatigue syndrome, 85 human immunodeficiency virus-infected patients, 50 with autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosis and 17 with leprosy, there was no positive case except one case each with alcohol addiction, AIDS, and dementia. Although 19 (1.36%) of 1,393 patients with various ocular diseases, 10 (1.09%) of 917 blood donors, and 3 (4.55%) of 66 multitransfused patients were seropositive for BDV-specific antigen, high levels of seroprevalence in schizophrenia patients and young patients (16 to 59 years old) with mood disorders were statistically significant. The immunoreactivity of seropositive sera could be verified for specificity by blocking with soluble p40 and/or p24 recombinant protein. Anti-p24 antibody was more frequent than p40 antibody in most cases, and in some psychotic patients antibody profiles showed only p40 antibody. Although serum positive for both p40 and p24 antibodies was not found in this study, the p40 ECLIA count in schizophrenia patients was higher than that of blood donors. Furthermore, we examined 90 sera from Japanese feral horses. Antibody profiles of control human samples are similar to that of naturally BDV-infected feral horses. We concluded that BDV infection was associated in some way with psychiatric disorders.

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ABSTRACT. Anti-Borna disease virus (BDV) antibodies were detected from cats in Japan, Philippines and Indonesia by using electrochemiluminescence immunoassay. Positive rates were 3.1%, 3.8% and 2.0% in Japan, Philippines and Indonesia, respectively. There was no differences in the positive rate of anti-BDV antibodies between male and female cats and among habitats. While, a significantly (P<0.05) higher prevalence (6.5%) was found in the oldest age group (more than 6 years) cats. KEY WORDS: Borna disease virus (BDV), electrochemiluminescence immunoassay (ECLIA), feline.J. Vet. Med. Sci. 63(8): 921-923, 2001 Borna disease is an infectious neurological disease that occurs sporadically in horses and sheep in Central Europe. Borna disease virus (BDV) is a noncytolytic, neurotropic, single strand, negative sense RNA virus that naturally infects a wide range of vertebrate species from birds and rodents to primates [1,3]. BDV can also cause encephalomyelitis in a wide range of experimental animals [5,6,14].In domestic cats, an characteristic disease of the central nervous system has been observed in Sweden [9]; the dominating clinical sign is an unsteady (staggering) gait. Histopathological examination of the cats showed that the inflammatory reaction of the central nervous system was most pronounced in the grey matter of the brain stem, basal ganglia and hippocampus. These changes and laboratory findings including leukopenia and elevations in protein content and white blood cell count of the cerebrospinal fluid in affected cats suggested that staggering disease is infectious and most probably caused by virus. By using an indirect immunofluorescence assay (IFA), anti-BDV antibody has been detected in the cats with typical staggering disease [10,13]. Recently, the high prevalence of BDV infections has been demonstrated in domestic cats with neurological disorders in Japan [11].A seroepidemiological survey of the cat has been done by using IFA and/or Western blot (WB) analysis also in Japan [12]. Since the antibody titers were usually very low in Borna disease, an establishment of sensitive and reliable method to detect a small amount of anti-BDV antibodies is required. Recently, we have developed a new system, electrochemiluminescence immunoassay (ECLIA), using recombinant BDV p24 and p40 proteins, which has the sensitivity and specificity to serve as a serological screening method for measuring anti-BDV antibodies [16]. In this study we tried to detect anti-BDV antibodies of domestic cats in Asian countries and to compare the prevalence of BDV among the countries.Blood samples were collected from domestic cats at veterinary hospitals of University of Philippines Los Baños, Laguna, Philippines; Faculty of Veterinary Science, Bogor Agricultural University, Bogor, Indonesia; Osaka, Kitakyushu, Kumamoto and Nagasaki, Japan. Serum samples were stored at -30°C until use.To measure antibody against BDV, ECLIA is performed as previously described [16]. Briefly, micro beads were coated with horse-derived BDV p24 and p40 reco...

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