Development of PCR-ELISA for the detection of hepatitis B virus x gene expression and clinical application.
The presence of hepatitis B virus x (HBx) antigen/antibody is known to correlate with the well-established serological markers of ongoing viral replication in the chronic phase of HBV infection, and strongly suggests that the level and duration of HBx expression may influence the outcome of the chronic infection. In this research, we developed a polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) method for the detection of HBx gene expression. We also investigated its relationship to the progress of the disease in HBV-related patients. Peripheral blood mononuclear cells (PBMCs) were purely isolated, and reverse transcription-PCR (RT-PCR) was performed for improved sensitivity. The PCR products were determined by ELISA, and we investigated the relationship of the proposed method to the clinical status of the patients. The PCR-ELISA used in this work was found to be at least 100 times more sensitive than the conventional PCR method, and even 8,000-fold diluted PCR products could be detected. The HBx concentrations significantly differed among control subjects (0.36+/-0.09, [P<0.01] and patients with chronic hepatitis (1.13+/-0.34 [P<0.01 compared to control]), liver cirrhosis (LC; 1.37+/-0.28 [P<0.01 compared to control]), and hepatocellular carcinoma (HCC; 1.48+/-0.95 [P<0.01 compared to control]). These findings suggest that monitoring of HBx could be useful for early diagnosis and prognosis in patients with chronic HBV infection, LC, and HCC.
Kim
Jong-Wan
JW
Department of Laboratory Medicine, College of Medicine, Dankook University, Cheon-An, South Korea.
Shim
Jung-Hyun
JH
Park
Joo-Won
JW
Jang
Won-Cheol
WC
Chang
H K
HK
Song
Il Han
IH
Baek
Sun-Young
SY
Lee
Seok-Ho
SH
Yoon
Do-Young
DY
Park
Sue-Nie
SN
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015967:Gene Expression Regulation, Viral; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein