Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

BACKGROUND: Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". METHODS: Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. RESULTS: The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. CONCLUSIONS: According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.

Correlation between serum hepatitis B virus core-related antigen and intrahepatic covalently closed circular DNA in chronic hepatitis B patients.

Nucleos(t)ide analogues are utilized for the treatment of chronic HBV infection, and HBe seroconversion and HBV DNA levels are commonly used as markers of viral status and as primary treatment endpoints. Recently, a new assay was prepared for the detection of serum HBV core-related antigen (HBcrAg), consisting of HBcAg, HBeAg, and p22cr, which is a precore protein from amino acid -28 to at least amino acid 150, by coding the precore/core region. In this study, we examined the correlation between serum HBcrAg concentration and viral status by the analysis of serum HBeAg, HBsAg, peripheral HBV DNA, and intrahepatic covalently closed circular DNA (cccDNA) in 57 chronic hepatitis B patients. Intrahepatic cccDNA was detected in all 57 patients, 42 patients were HBcrAg-positive, and serum HBcrAg concentration level was closely correlated with cccDNA. Additionally, positive HBcrAg concentration level results were observed in 6 out of 13 HBsAg seroclearance patients and 20 out of 31 HBV DNA-negative patients. Moreover, the correlation between HBcrAg and cccDNA in these 31 HBV DNA-negative patients was statistically significant (r = 0.482, P = 0.006). These data suggest that serum HBcrAg concentration is well correlated with intrahepatic cccDNA level, and that the measurement of serum HBcrAg may be clinically useful for monitoring intrahepatic HBV viral status, especially in patients under treatment with nucleos(t)ide analogues.

Performance of ELISA for specific measurement of high-molecular-weight (HMW) adiponectin.

Adiponectin is an adipocyte-specific protein secreted as trimer, hexamer and high-molecular-weight (HMW) complex. Several reports have indicated that the biologically active form of adiponectin is HMW, and that HMW adiponectin concentration is correlated with insulin sensitivity and metabolic disorders including diabetes better than total adiponectin concentration. We developed a sandwich ELISA using a monoclonal antibody against HMW adiponectin purified from human serum. The specificity of established ELISA to HMW adiponectin was confirmed by analysis of human serum fractions prepared by gel filtration chromatography. After careful purification of HMW adiponectin for the calibration standard, the concentration was determined by the amino acid analysis, protein concentration assay by Lowry's method and theoretical calculation from amino acid sequence of adiponectin. An appropriate calibration curve was obtained by HMW adiponectin standards and an assay range is from 0.2 to 25 ng/mL. Intra- and inter-assay coefficient of variation (CV) was below 3.0% and 5.1%, respectively. The recovery of added HMW adiponectin was from 98.5% to 99.2%. No interference of blood components was confirmed by adding free and conjugated forms of bilirubin, hemoglobin and lipids. Serum is suitable as samples and could be stored at 10 degrees C in the refrigerator for at least 28 days stably. Our HMW adiponectin-specific ELISA system is simple, without any procedure of sample pretreatment, reliable, suitable for routine analysis and highly useful for elucidating the clinical significance of adiponectin.

Analytical evaluation of a high-molecular-weight (HMW) adiponectin chemiluminescent enzyme immunoassay.

BACKGROUND: The measurement of high-molecular-weight (HMW) adiponectin concentration provides valuable clinical information. However, the conventional ELISA method requires complicated and lengthy assay procedures to obtain assay results. METHODS: We prepared new assay reagents based on chemiluminescent enzyme immunoassay (CLEIA) on a fully-automated analyzer system using the same IH7 monoclonal antibody as for ELISA as solid phase and detection antibodies (CLEIA/cartridge-type and CLEIA/bottle-type). RESULTS: The assay range of both CLEIA reagents were from 0.20 to 15.00 µg/ml, and lower limit of detection and quantification were lower than 0.0928 and 0.1346 µg/ml in CLEIA/cartridge-type and in CLEIA/bottle-type reagents, respectively. A good correlation was observed between both reagents (y = 1.000x + 0.120). The imprecision test as % of coefficient variation in both reagents were less than 3.3% and recovery test showed the range from 100% to 109%. No or little interference of blood components was observed in both reagents. HMW adiponectin concentration measured by CLEIA reagents was approximately half that measured by the previous ELISA because of reevaluation using freshly and highly purified HMW adiponectin standard. CONCLUSION: The newly prepared CLEIA reagents are robust and adequate and can be used for the measurement of HMW adiponectin in the clinical laboratory.