[Evaluation and application of a newly developed highly sensitive HBsAg chemiluminescent enzyme immunoassay for chronic hepatitis B patients].

AIM: A high sensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for quantitative hepatitis B surface antigen (HBsAg) detection by a combination of monoclonal antibodies, each one for a specific epitope of HBsAg, and by improving the conjugation technique (Matsubara, et al. Transfusion 2009). We modified and automated Matsubara's techniques. Our aim is to evaluate the fundamental performance of our assay (prototype) and to investigate the clinical significance of prototype in patients with hepatitis B virus (HBV) infection. METHODS: We used 226 HBsAg-negative samples and 59 HBsAg-positive samples for evaluation of prototype's accuracy, reproducibility, specificity and sensitivity. We examine correlation between the prototype assay and commercial quantitative HBsAg detection assay (the Abbott ARCHITECT). Performance of prototype was compared with the Abbott ARCHITECT in one chronic hepatitis B patient and one patient with HBsAg seroconversion sequentially. RESULTS: The prototype assay had good accuracy, reproducibility, specificity and sensitivity. There is positive correlation between the prototype and the Abbott ARCHITECT. The sensitivity of the prototype (5 mIU/mL) was approximately 10 fold higher than the Abbott ARCHITECT (50 mIU/ml). The prototype could detect HBsAg at the HBsAg-negative point by Abbott ARCHITECT in these patients. CONCLUSIONS: Automatic highly sensitive HBsAg CLEIA prototype is convenient and precise assay for HBV monitoring.

[Evaluation of high-sensitivity HBsAg quantitative assay for HBV genotype].

The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations has been reported in HBV-infected patients during anti-viral treatment. HBV genotypes A and D are ubiquitous and scattered worldwide, especially northern America as well as Europe, whereas genotypes B and C are common in Asia. The aim of this study was to evaluate a new version of the Sysmex HBsAg quantitative kit based on Chemiluminescence Enzyme Immunoassay. Sera collected from 172 patients infected with any of the four major genotypes A to D (HBV/A, n = 18; B, n = 25; C, n = 84; D, n = 45), including the genotype D cases with weak reaction in the previous version of the kit. The new version of the kit having additional monoclonal antibody, showed improved sensitivity compared to the previous version as well as robust correlation with another quantitative HBsAg assay: the Abbot Architect. Observed during lamivudine therapy, increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for the four HBV genotypes common worldwide. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response or resistance to anti-viral therapy.

[Evaluation of hepatitis B virus genotyping EIA kit].

Clinical significance of Hepatitis B virus(HBV) genotyping is increasingly recognized. The aim of this study was to evaluate reproducibility, accuracy, and sensitivity of an enzyme immunoassay (EIA) based HBV genotyping kit, which designed to discriminate between genotypes to A, B, C, or D by detecting genotype-specific epitopes in PreS2 region. Using the four genotypes panels, the EIA demonstrated complete inter and intra-assay genotyping reproducibility. Serum specimens had stable results after 8 days at 4 degrees C, or 10 cycles of freezing-thawing. In 91 samples that have been genotyped by DNA sequencing, 87(95.6%) were in complete accordance with EIA genotyping. Of examined 344 HBsAg-positive serum specimens, genotypes A, B, C and D were determined in 26 (7.6%), 62 (18.0%), 228 (66.3%), and 9 (2.6%) cases, respectively. Of 19 (5.5%) specimens unclassified by the EIA, 13 were found to have low titer of HBsAg concentration (< 3 IU/ml), and the other 5 had amino acid mutations or deletions within targeted PreS2 epitopes. The EIA allowed genotyping even in HBV DNA negative samples (96.2%). In conclusion, HBV genotype EIA is reliable, sensitive and easy assay for HBV genotyping. The assay would be useful for clinical use.

[Clinical evaluation of a novel HBsAg quantitative assay].

The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations in HBV-infected individuals remains unclear. The aim of this study was to evaluate a novel fully automated Chemiluminescence Enzyme Immunoassay (Sysmex HBsAg quantitative assay) by comparative measurements of the reference serum samples versus two independent commercial assays (Lumipulse f or Architect HBsAg QT). Furthermore, clinical usefulness was assessed for monitoring of the serum HBsAg levels during antiviral therapy. A dilution test using 5 reference-serum samples showed linear correlation curve in range from 0.03 to 2,360 IU/ml. The HBsAg was measured in total of 400 serum samples and 99.8% had consistent results between Sysmex and Lumipulse f. Additionally, a positive linear correlation was observed between Sysmex and Architect. To compare the Architect and Sysmex, both methods were applied to quantify the HBsAg in serum samples with different HBV genotypes/subgenotypes, as well as in serum contained HBV vaccine escape mutants (126S, 145R). Correlation between the methods was observed in results for escape mutants and common genotypes (A, B, C) in Japan. Observed during lamivudine therapy, an increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for all HBV genetic variants common in Japan. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response to lamivudine treatment and diagnosis of the breakthrough hepatitis.

[Fundamental and clinical evaluation of hepatitis B virus core-related antigen assay by LUMIPULSE f].

A sensitive chemiluminescence enzyme immunoassay (CLEIA) has been developed for hepatitis B virus (HBV) core-related antigens (HBcrAg) detection. The HBcrAg is designated as the precore/core gene products including HBeAg. The aim of this study is to evaluate reproducibility of HBcrAg and correlation with HBV-DNA in serum using the automatic LUMIPULSE f to estimate an assay suitable for general laboratory use. In this study, we demonstrated that HBcrAg assay had highly intra-assay reproducible [coefficients of variation(CVs); 2.8-5.2%] and inter-assay reproducible [CVs; 3.9-9.1%]. When the cutoff value was tentatively set at 1 kU/ml, all healthy controls (HBsAg/HBV-DNA negative; n=100) and anti-HCV antibody-positive (n=50) sera were identified as negative. The assay showed a detection limit of 0.5 kU/ml using four serially diluted HBV high-titer sera, indicating higher sensitivity than HBV-DNA (transcription-mediated amplification). The HBcrAg concentration correlated positively with serum HBV-DNA (n=125, r = 0.860, p < 0.0001) regardless of HBeAg, although the HBcrAg levels were higher in HBeAg-positive group than in HBeAg-negative group. In the natural course of HBV infection, the HBcrAg concentration usually changed in accordance with HBV-DNA levels, however during lamivudine therapy the change of HBcrAg was more gradual than that of HBV-DNA. In conclusion, 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.