Antibodies to the E2 protein of GB virus C/hepatitis G virus: low prevalence in Asian countries.

Epidemiological investigations of GB virus C (GBV-C)/hepatitis G virus (HGV), an infectious agent discovered in 1995/1996, are facilitated by a recently developed immunoassay for the detection of antibodies to the viral envelope 2 protein (anti-E2). We used this assay to establish GBV-C/HGV prevalence in seven European, African, and Asian countries. A total of 1579 serum samples from healthy adults lacking prior exposure to known parenteral risk factors was screened. Anti-E2 positivity ranged from 13.6% (Italy) to 7.7% (Mauritius) in the European and African countries investigated. Anti-E2 prevalence was exceedingly low in the Philippines and Sri Lanka. This observation might be attributable to socio-economical and demographic factors.

Identification of hepatitis G virus particles in human serum by E2-specific monoclonal antibodies generated by DNA immunization.

In order to elucidate the structure and morphology of hepatitis G virus (HGV), a recently isolated flavivirus, we generated a panel of eight monoclonal antibodies (MAbs) against the putative second envelope protein (E2) following DNA immunization. The MAbs were shown to be specific for four different epitopes on recombinant E2. MAb Mc6 was the only antibody able to detect the linear epitope LTGGFYEPL. In addition, Mc6 was able to immunoprecipitate viral particles in human blood samples as detected by reverse transcription-PCR amplification of HGV RNA. This precipitation could be competed by addition of saturating amounts of the linear peptide or abolished by addition of Nonidet P-40. We conclude that, albeit lacking the N-terminal sequence of a functional core protein, HGV builds classical viral particles displaying E2 envelope protein on their outer surfaces.

Distinct prevalence of antibodies to the E2 protein of GB virus C/hepatitis G virus in different parts of the world.

Since the identification of the new human virus, GB virus C (GBV-C)/hepatitis G-virus (HGV), in 1995/1996, reverse transcription polymerase chain reaction remained the sole available diagnostic tool for GBV-C/HGV infection. Recently, a serologic test based on the detection of antibodies to the putative envelope protein 2 (anti-E2) has been introduced. We used this assay for a seroepidemiological survey including 3,314 healthy individuals from different parts of the world, 123 patients from Germany who were suspected to have an increased risk of acquiring GBV-C/HGV infection, 128 multiple organ donors, and 90 GBV-C/HGV RNA positive persons. In European countries, anti-E2 seropositivity ranged from 10.9% (Germany) to 15.3% (Austria). In South Africa (20.3%) and Brazil (19.5%), even higher anti-E2 prevalence rates were recorded. In Asian countries like Bhutan (3.9%), Malaysia (6.3%), and the Philippines (2.7%), anti-E2 positivity was significantly lower. GBV-C/HGV anti-E2 prevalence in potential "risk groups," i.e., patients on hemodialysis and renal transplant recipients, did not vary significantly from anti-E2 seroprevalence in German blood donors. Anti-E2 and GBV-C/HGV RNA were found to be mutually exclusive, confirming the notion that anti-E2 has to be considered as a marker of past infection.

Humoral immune response to the E2 protein of hepatitis G virus is associated with long-term recovery from infection and reveals a high frequency of hepatitis G virus exposure among healthy blood donors.

The second envelope protein (E2) of the hepatitis G virus (HGV) was expressed in Chinese hamster ovary (CHO) cells and showed a molecular weight of approximately 60 to 70 kd, with 15 to 25 kd of the size contributed by N-linked glycosylation. An enzyme-linked immunosorbent assay (ELISA) using HGV-E2 was developed to test for antibodies to this protein (anti-E2) in human sera. High sensitivity was achieved by developing monoclonal antibodies (mAbs) to HGV-E2, which were used as capture antibodies in the ELISA. Our studies revealed that 16% of healthy Spanish blood donors were exposed to HGV, indicating that additional routes of viral transmission besides parenteral exposure might exist. An even higher prevalence of exposure to HGV (52%-73%) was found in several groups at risk of parenteral exposure to infectious agents, i.e., intravenous drug users, transfusion history, hemophiliacs, and hepatitis C virus (HCV)-positive patients. Most anti-E2-positive patients were HGV-RNA-negative and vice versa, indicating an inverse correlation of these two viral markers. A panel of 16 posttransfusion patients followed for up to 16 years revealed that patients who develop an anti-E2 response become HGV-RNA-negative, while patients who do not develop anti-E2 are persistently infected. Immunity to HGV seems to be long-lasting, because circulating antibody to E2 could still be detected 14 years after seroconversion. Sequence comparisons showed that E2 is highly conserved among isolates collected worldwide, indicating that immune escape variants are not common in HGV infections. This reflects on a molecular level why HGV infections usually are cleared spontaneously by the host. However, possible mechanisms of HGV persistence, as found in some patients, remain to be elucidated.

Concentration measurement of unpurified proteins using biosensor technology under conditions of partial mass transport limitation.

Using biosensor technology, it is possible to measure protein concentration when the binding of the protein to an appropriate ligand immobilized on the sensor surface is totally limited by diffusion and mass transport, a condition difficult to achieve in practice. In such a case, the observed binding rate does not reflect the intrinsic binding capacity of the molecular partners, but is simply proportional to the concentration of the protein analyte that is introduced in a continuous flow over the ligand. We describe here a more general biosensor method for measuring protein concentration which is applicable to conditions where mass transport is not totally but only partially rate limiting. The proposed method, which is based on measurements at different flow rates, does not require a standard of known protein concentration and can be used with unpurified proteins. The method is applicable to ligand-analyte pairs with an association rate constant as low as 10(3) M-1 s-1 and requires only knowledge of the molecular weight and diffusion coefficient of the analyte. The method was used successfully to measure the concentration of monoclonal antibodies, monoclonal antibody fragments (Fab) obtained by papain cleavage, and recombinant Fab fragments of widely different affinities in crude Escherichia coli extracts.

Detection of antibodies to a putative hepatitis G virus envelope protein.

BACKGROUND: A flavivirus designated hepatitis G virus (HGV) has been isolated from the serum of patients with non-A-E hepatitis. Hitherto, the presence of HGV RNA in serum has been detected with the reverse transcription-polymerase chain reaction (RT-PCR) amplification method. We have now developed an immunoassay for antibodies against an HGV protein. METHODS: Recombinant HGV envelope protein E2 was used as antigen in an ELISA. 80 blood donors, 99 intravenous-drug users, and 11 patients with acute post-transfusion hepatitis were tested for antibodies to E2. The HGV-RNA status was assessed by RT-PCR. FINDINGS: Anti-E2 seroprevalence was 9% among the blood donors and 41% among the drug users; HGV-RNA prevalence was 2.5% and 38%, respectively. Whereas anti-E2 prevalence increased with the duration of drug use, HGV-RNA prevalence declined in parallel. In each group, the presence of anti-E2 and HGV RNA was almost mutually exclusive: none of the blood donors and only 4% of the drug users were positive for both markers at the same time. Of the 11 post-transfusion patients--who were all HGV-RNA positive and anti-E2 negative at the onset of disease--four developed antibodies to E2 during the following year, and two of the four subsequently became HGV-RNA negative. INTERPRETATION: We conclude that a humoral immune response to E2 is associated with loss of detectable HGV viraemia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. The immunoassay we describe should facilitate investigation of suspected infections and may be helpful in the elucidation of the clinical significance of HGV.

Reverse transcription-PCR detection of hepatitis G virus.

Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.

A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo.

A novel sandwich immunoassay for measurement of soluble fibrin in plasma has been developed. For immunization we used the synthetic heptapeptide Gly-Pro-Arg-Val-Val-Glu-Arg representing the amino terminus of the alpha-chain of human fibrin. A monoclonal IgG1 antibody was obtained by conventional hybridoma technology. To increase convenience, the sandwich immunoassay was developed for the Enzymun-Test systems which are based on streptavidin pre-coated tubes. The new assay was designed with the same fibrin specific antibody both in biotinylated and in peroxidase-labelled form. Fibrin in native plasma samples could only be detected after pre-incubation of the plasma with chaotropic ions. Test results were calculated using a standard curve comprising six fibrin standards (0-50 micrograms/ml). Precision of the method was satisfactory; intra-assay CVs using plasma samples ranged between 5.0% (23.7 micrograms/ml) and 12.4% (0.2 microgram/ml). CVs of interassay precision measurements using standards as samples range between 7.3% (25.0 micrograms/ml) and 11.4% (1.0 micrograms/ml). The lower detection limit was 0.12 microgram/ml. Investigations of normal range in 70 age-matched healthy individuals resulted in a mean of 1.12 micrograms/ml. Linearity was excellent; recovery of high fibrin plasma after dilution with normal plasma was always between 100 and 107%. Fibrin specificity was due to the monoclonal antibody 2B5 used and no cross reactivity with fibrinogen, fibrinogen split products or fibrin D-dimer was observed. Fibrin fragment E1 (studied by Dempfle CE, et al. Blood Coag Fibrinol 1993; 4: 79-86) and fragments X and Y showed moderate cross reactivity in the assay and caused some overestimation of fibrin at high fibrin split product concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)