Digene® HBV Test Hybrid Capture® II

A Signal Amplification Hybridization Microplate Test for the Chemiluminescent Detection and Quantitation of Hepatitis B Virus (HBV) DNA in Serum

  SUMMARY AND EXPLANATION

Hepatitis B virus (HBV) is one of the causative agents of viral hepatitis. There are an estimated 300 million chronic carriers of HBV worldwide. Over 50% of people infected develop symptoms of acute disease, such as jaundice, nausea, anorexia, malaise or fatigue. Progression to chronic disease, which occurs in 5% of adults and up to 90% of newborns is of great public health concern. Up to 25% of chronic carriers subsequently suffer cirrhosis and liver failure or develop hepatocellular carcinoma (HCC) (1). HBV is found in the blood and other body fluids and can be transmitted sexually, during childbirth, through the use of subcutaneous needles, or during liver transplantations. Both chronic and acutely infected patients are capable of transmitting HBV (2, 3, 4).

HBV is composed of a 27 nm nucleocapsid which is surrounded by a 42 nm surface envelope. The surface envelope carries the hepatitis B surface antigen (HBsAg), the main serological marker for HBV. It may exist as an envelope surrounding the nucleocapsid or alone as a 22 nm noninfectious proteinaceous
particle (5).

Several forms of hepatitis B prophylaxis and treatment are currently available. Active immunization with recombinant vaccines or passive immunization through injection of pooled serum containing high titers of anti-HBs are effective in protecting from HBV infection. Treatments for chronic hepatitis B include ∞-interferon, to which about 20% of patients respond favorably, and lamivudine, a nucleoside analog that has recently shown great promise by drastically reducing viral load in nearly all treated chronic hepatitis B patients (6, 7, 8).

Because of the potentially severe consequences of acute and chronic HBV infection, it is important to be able to monitor the disease’s progression closely. Studies have shown that detectable HBV DNA levels persisting longer than eight weeks may be indicative of progression to chronic hepatitis B (9, 10), thus the ability to detect HBV DNA in serum has prognostic value for the outcome of acute hepatitis B infection.
Serological markers and DNA tests are routinely used as diagnostic indicators of acute and chronic HBV infection. However, serological markers often do not reflect the disease’s true progression (4, 11). The presence of HBV DNA in serum is an accurate marker of viral replication. With the advent of new potent nucleoside analog drugs it has become increasingly important to monitor very low levels (less than 104 copies/ml serum) of HBV DNA. Hence, a highly sensitive quantitative test for HBV DNA is required for monitoring progression of chronic HBV infection.

  REAGENTS AND MATERIALS PROVIDED

The Digene® HBV Test using Hybrid Capture® II technology is a signal amplification hybridization antibody capture microplate test that utilizes chemiluminescent detection. Specimens containing the target DNA hybridize with an RNA probe mix specific to HBV ad and ay strains. The resultant RNA:DNA hybrids are captured onto the surface of microplate wells coated with antibodies specific for RNA:DNA hybrids. Immobilized hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA:DNA hybrids. Several alkaline phosphatase molecules are conjugated to each antibody and multiple conjugated antibodies bind to each captured hybrid resulting in substantial signal amplification. The bound alkaline phosphatase then cleaves a chemiluminescent substrate. The emitted light is measured as relative light units (RLUs) on a luminometer. A calibration curve of calibrators is plotted and the RLU value of each specimen is compared to that of the curve to determine the concentration of HBV DNA in the test specimens. If very low levels of HBV DNA are to be quantitated, the virions can be concentrated by centrifugation before the test is performed.