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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
Reagents for 96 Tests
1 use: up to 82 specimens
2 uses: up to 34 specimens each
3 uses: up to 18 specimens each
4 uses: up to 10 specimens each
THIS KIT CONTAINS BLOOD PRODUCTS AND INFECTIOUS MATERIALS. HANDLE
MATERIAL IN COMPLIANCE WITH YOUR LABORATORY REQUIREMENTS AND YOUR
LOCAL AND FEDERAL STANDARDS.
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.
PRINCIPLE OF THE PROCEDURE
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.
REAGENTS AND MATERIALS PROVIDED
Two formats of the Digene® HBV Test are available; the Standard
format and the Ultra-Sensitive format. Below are the catalog
numbers and reagents materials provided in each format.
Catalog Number: 6110-1096 RUO HBV HCII Standard Test 96 Tests
(10, 18, 34 or 82 specimens)
1 x 0.15 ml Positive Control-2 Contains infectious HBV virons and
sodium azide
1 x 0.15 ml Positive Control-3 Contains infectious HBV virons and
sodium azide
1 x 0.35 ml Indicator Dye: Contains sodium azide
1 x 4.5 ml Denaturation Reagent: Dilute sodium hydroxide (NaOH)
solution
1 x 6.5 ml Probe Diluent: Buffered solution with sodium azide
1 x 250 l HBV Probe: HBV RNA probes (ad and ay subtypes) in
buffered solution
1 x 0.4 ml Calibrator 1: Carrier DNA in HBV negative human serum
with sodium azide
1 x 0.4 ml Calibrator 2: 1.42 x 105 copies/ml (0.5 pg/ml) HBV
plasmid DNA and carrier DNA in HBV negative human serum with
sodium azide
1 x 0.3 ml Calibrator 3: 2.83 x 107 copies/ml (100 pg/ml) HBV
plasmid DNA and carrier DNA in HBV
negative human serum with sodium azide
1 x 0.3 ml Calibrator 4: 5.66 x 108 copies/ml (2000 pg/ml) HBV
plasmid DNA and carrier DNA in HBV negative human serum with
sodium azide
1 x 0.3 ml Calibrator 5: 1.70 x 109 copies/ml (6000 pg/ml) HBV
plasmid DNA and carrier DNA in HBV negative human serum with
sodium azide
1 each Capture Microplate: Microtiter plate coated with anti-RNA:DNA
hybrid antibodies
1 x 8 ml Detection Reagent 1: Alkaline phosphatase-conjugated
antibodies to RNA:DNA hybrids in buffered solution with sodium
azide
1 x 8 ml Detection Reagent 2: CDP-Star™ with Emerald II (chemiluminescent
substrate)
1 x 100 ml Wash Buffer Concentrate: Contains sodium azide
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