Highly Sensitive Detection of Hepatitis B Virus Surface Antigen by Use of a Semiautomated Immune Complex Transfer Chemiluminescence Enzyme Immunoassay

Abstract

The performance of hepatitis B surface antigen (HBsAg) screening assays is continuously improved to reduce the risk of transfusion-associated hepatitis B. In this study, a semiautomated immune complex transfer chemiluminescence enzyme immunoassay (ICT-CLEIA) for the detection of HBsAg, which is as sensitive as hepatitis B virus (HBV) DNA PCR, was developed; the ICT-CLEIA assay performance was compared with the performance of the Architect HBsAg QT assay and HBV DNA PCR. The specificities in the initial assay and after retesting were 99.50% (1,988/1,998 samples) and 99.95% (1,997/1,998 samples), respectively. The analytical detection limit was determined to be 0.2 mIU/ml using the 2nd International WHO HBsAg standard, and the cutoff value (0.5 mIU/ml) of the ICT-CLEIA assay was 8.0 standard deviations (SD) above the mean of the HBsAg-negative specimens. The ICT-CLEIA assay could detect HBsAg even in the presence of anti-HBs antibodies and demonstrated a 23.6-dayshorter window period using commercially available HBsAg seroconversion panels than the Architect HBsAg QT assay. Furthermore, the monitoring of the viral kinetics by the ICT-CLEIA assay and the HBV DNA PCR produced very similarly shaped curves during both the HBsAg seroconversion and reverse seroconversion periods. Therefore, the ICT-CLEIA assay may be useful not only for an earlier detection of HBV reactivation but also for the monitoring of hepatitis B patients.

A novel general and efficient technique for dissociating antigen in circulating immune complexes

“…In the dissociation, the isotonic state was assured during the dissociation, neutralization and measurement, which assures that the antigens are not influenced by the measurement. This has never been reported in previous studies . In addition, glycine‐HCl was found as the best buffer system.…”

“…The tolerance of antigens in different CICs to pH is different. Thus, the traditional HCl dissociation method, trypsin digestion method and Immune Complex Transfer method are only used in the measurement after dissociation of HBsAg‐IC, HCV Core‐IC and HIV P24‐IC . However, the values determined with these methods were also lower than those determined with our new method.…”

“…HCl Dissociation , Trypsin Digestion and Immune Complex Transfer (three methods used for the detection of CICs with specific antigen) as well as our technique were employed to separate, dissociate or trypsinize HBsAg‐IC (five HBV‐M patterns; n = 335), HCV core‐IC ( n = 32), HIV P24‐IC ( n = 12), Ins‐IC ( n = 35) and TG‐IC ( n = 56). Different antigens were detected using ELISA HCV core antigen assay kit, ELISA HIV P24 antigen assay kit and ELISA‐anti‐Clq capture assay kit.…”

“…Immune complexes were degraded by immune complex transfer method of Takeda K et al. (2013) with slight modification. In short, the above sediment tubes (test and control; n = 2) was mixed with 150 μL 0.9% NaCl, followed by addition of 100 μL of pretreatment reagent 1 (15 mM phosphate buffer containing 0.15 N NaOH, 1.2 M urea, and 0.4% Brij35) or 0.9% NaCl.…”

Longitudinal profiles of highly sensitive hepatitis B surface antigen levels: re‐evaluation of HBsAg seroclearance

“…Positive results were confirmed with anti‐HBs neutralizing antibodies. The lower detection limit of the hs‐HBsAg assay was 0.5 mIU/ml .…”