Tatsuji Kimura

Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load

ABSTRACT A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 103 U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 × 102 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

Increased Plasma S100A12 (EN-RAGE) Levels in Hemodialysis Patients with Atherosclerosis

Background: S100A12, also known as EN-RAGE (extracellular newly identified receptor for advanced glycation end products binding protein) is a ligand for RAGE, and has been proposed to contribute to the development of atherosclerosis. In this study, we examined the plasma S100A12 concentration in patients with ESRD and undergoing hemodialysis (HD) and evaluated the relation between S100A12 level and carotid intimal media thickness (IMT) by ultrasound. Methods: We measured plasma S100A12 concentration in 72 HD patients and 42 control subjects. IMT of the carotid artery was measured by high-resolution B-mode ultrasonography in 46 HD patients. Results: The mean plasma S100A12 level was 2.3-fold higher in HD patients than in control subjects (25.0 ± 2.32 vs. 10.7 ± 0.97 ng/ml, p < 0.001). Stepwise multiple regression analysis identified circulating white blood cell count as a positive independent determinant and total cholesterol and serum albumin levels as negative independent determinants of plasma S100A12 concentration. The maximum IMT was positively correlated with plasma S100A12 level. Stepwise multiple regression analysis also identified plasma S100A12 as a significant independent determinant of the maximum IMT. Conclusion: These findings suggest that S100A12 protein is involved in the acceleration of atherosclerosis in HD patients.

Clinical evaluation of a new enzyme immunoassay for hepatitis B virus core‐related antigen; a marker distinct from viral DNA for monitoring lamivudine treatment

Summary. We aimed to assess the clinical performance of a newly developed chemiluminescence enzyme immunoassay (CLEIA) for the detection of hepatitis B virus (HBV) core‐related antigen (HBcrAg) in patients with chronic HBV infection. A total of 82 patients with chronic HBV infection and 167 HBV‐negative controls were studied. HBcrAg was measured by CLEIA with monoclonal antibodies to hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg), and HBV DNA was measured by transcription‐mediated amplification assay (TMA) and in‐house real‐time detection polymerase chain reaction (RTD‐PCR). The HBcrAg assay detected viremia in 189 of 216 samples (88%) collected from 72 patients whilst the TMA assay detected viremia in 178 of the 216 samples (82%) (P = 0.019). The HBcrAg concentration correlated linearly with the HBV DNA concentration (P < 0.001) over a range which varied 100 000‐fold. The accuracy in the measurement of the patients’ HBV load obtained using the HBcrAg assay was not affected by the absence of hepatitis B e antigen from the serum or the presence of precore mutations in the HBV genome. In patients without anti‐viral drugs, changes in their serum HBcrAg concentration over time corresponded to their HBV DNA concentration. In six additional patients who were later treated with lamivudine, HBV DNA concentration declined more rapidly than their HBcrAg concentration. Three months after treatment commenced, the ratio of HBcrAg: HBV DNA had increased in all six patients (P = 0.031). The HBcrAg assay is a sensitive and useful test for the assessment of a patients HBV load. When monitoring the anti‐viral effect of lamivudine, HBcrAg provides a viral marker which is independent of HBV DNA.

New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA

ABSTRACT A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 μg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.

Measurement of hepatitis B virus core-related antigen is valuable for identifying patients who are at low risk of lamivudine resistance

Abstract: Objective: The clinical usefulness of hepatitis B virus core‐related antigen (HBVcrAg) assay was compared with that of HBV DNA assay in predicting the occurrence of lamivudine resistance in patients with chronic hepatitis B.

Hepatitis B virus RNA is measurable in serum and can be a new marker for monitoring lamivudine therapy

BackgroundChanges in the serum hepatitis B virus (HBV) RNA level during lamivudine therapy were compared to those in the serum HBV DNA and HBV core-related antigen (HBVcrAg) levels in 24 patients with chronic hepatitis B.MethodsFor measurement of HBV RNA, total nucleic acid was extracted from serum samples and treated with RNase-free DNase I. After cDNA synthesis from extracted RNA, HBV RNA was measured by real-time detection polymerase chain reaction.ResultsThe peak fraction of HBV RNA in serum samples was consistent with peak fractions of HBV DNA and HBV core protein in a sucrose gradient analysis, indicating that HBV RNA was incorporated into virus particles. All levels of HBV DNA, HBV RNA, and HBVcrAg decreased gradually during lamivudine therapy (P < 0.001 for all). The amount of decrease from the start of lamivudine therapy was significantly higher for HBV DNA than for HBV RNA or HBVcrAg during 6 months of lamivudine therapy (P < 0.001 for all). However, a similar difference was not seen between HBV RNA and HBVcrAg levels during that period. The HBV RNA level was significantly correlated (P < 0.001 for all) with levels of HBV DNA and HBVcrAg both at the beginning and 2 months after the start of lamivudine therapy.ConclusionsHBV RNA is detectable in serum in a form indicating incorporation into virus particles, and its serum level might serve as a new viral marker with a significance different from that of HBV DNA in lamivudine therapy.

Plasma S100A12 Concentrations in Peritoneal Dialysis Patients and Subclinical Chronic Inflammatory Disease

Abstract:  S100A12 is a ligand for the receptor for advanced glycation end products. It has been shown that S100A12 induces expression of adhesion molecules, and mediates activation and migration of monocytes/macrophages. Circulating S100A12 may be involved in chronic inflammation. We previously reported increased S100A12 levels in patients with non‐insulin‐dependent diabetes mellitus and hemodialysis. A high peritoneal solute transport rate may be associated with encapsulating peritoneal sclerosis and mortality. We measured plasma S100A12 levels in peritoneal dialysis patients and evaluated a possible relation between the increased plasma S100A12 levels in peritoneal dialysis patients and the high peritoneal solute transport rate. Subjects included 36 patients (mean age ± SE, 46.0 ± 12.0 years) with no apparent infection and no malignancy who had been undergoing peritoneal dialysis for 36.5 ± 3.9 months. We developed an enzyme‐linked immunosorbent assay system to measure plasma S100A12 levels. A peritoneal equilibrium test was performed and subjects were categorized as high and high‐average (H) (n = 14) or low and low‐average (L) (n = 22) transporters. Plasma S100A12 concentrations were significantly higher in peritoneal dialysis patients (21.6 ± 3.0 ng/mL) than in control subjects (n = 42; 10.8 ± 1.0 ng/mL). Plasma S100A12 concentrations were also higher in the H group (28.2 ± 6.1 ng/mL) than in the L group (14.2 ± 2.0 ng/mL). These results suggest that S100A12 may be a sensitive marker of subclinical inflammation and that an increased S100A12 level may be related to the high peritoneal solute transport rate.

Hepatitis B virus core and core-related antigen quantitation in Chinese patients with chronic genotype B and C hepatitis B virus infection.

Background and Aims:  Hepatitis B virus (HBV) core‐related antigen (HBcrAg) and HBV core antigen (HBcAg) assays were developed for the measurement of serum HBV load. The aim of this study was to assess the clinical utility of these assays in Chinese patients with chronic genotype B and C HBV infection.

Development of specific and quantitative real-time detection PCR and immunoassays for λ3-interferon

Aim:  Single nucleotide polymorphisms (SNP) around interferon (IFN)‐λ3 have been associated with the response to pegylated IFN‐α treatment for chronic hepatitis C. Specific quantification methods for IFN‐λ3 are required to facilitate clinical and basic study.