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1
Overview of Commercial
HBV Assay Systems
Stefan Zeuzem
1. Introduction
Measurement of viral nucleic acid in serum is often a valuable adjunct to the man-
agement of viral infections (1). In hepatitis B, tests for hepatitis B virus (HBV) DNA
have been used widely (Table 1), but their interpretation and significance have yet to be
defined. HBVDNAassays are limited by lack of standardization and variable sensitiv-
ity. Because HBV may circulate in serum at high levels (as high as 1010 virions/mL),
direct molecular hybridization assays are capable of detecting HBVDNAin a high pro-
portion of patients, particularly those with active disease and both HBsAg (hepatitis B
surface antigen) and HBeAg (hepatitis B e antigen) in serum. Commercial assays com-
prise the liquid hybridization assay (Genostics™, Abbott Laboratories, Chicago, IL),
the hybridization capture assay (Digene, HC II), and branched DNA (bDNA) signal
amplification assay (Versant, Bayer Diagnostics). Furthermore, a quantitative poly-
merase chain reaction (PCR) assay for HBVDNAhas been developed (Amplicor Mon-
itor HBV, Roche Diagnostics); it detects HBVDNAin a higher proportion of patients
with chronic hepatitis B and often yields positive results, even in HBsAg carriers with-
out apparent disease.
2. HBVDNAQuantification Assays
2.1. Liquid Hybridization Assay
The Genostics HBV DNA assay was a liquid-phase molecular hybridization assay
(Fig. 1A) that involved the hybridization of HBV genomic DNA to single-stranded
125I-DNA probes in solution (2,3). A sepharose column was used to separate the base-
paired HBVDNAfrom the excess single-stranded 125I-DNA, and the radioactivity in the
column eluate was measured in a gamma counter. The radioactivity in each specimen was
compared with that of positive and negative control standards, and results were expressed
as picograms per milliliter (pg/mL). The test required 100 (cid:1)Lof serum for a single deter-
From: Methods in Molecular Medicine, vol. 95: Hepatitis B and D Protocols, volume 1
Edited by: R.K. Hamatake and J.Y.N. Lau © Humana Press Inc., Totowa, NJ
3
4 Zeuzem
Table 1
Different Principles of HBVDNAQuantification
Signal amplification assays
Liquid hybridization
DNA–RNAhybridization
Branched DNAtechnology, bDNA
Target amplification assays
Polymerase chain reaction (PCR)
Transcription-mediated amplification (TMA)
Nucleic acid based amplification (NASBA)
Ligase chain reaction (LCR)
mination. The positive control standard included in the assay consisted of M13 phage con-
taining the 3.2 kb HBVDNAgenome (-) strand, quantified by plaque assays and diluted
into HBV-negative human serum to a final concentration of 103 ±10 pg DNA/mL(2,4).
The assay was applied in many clinical trials. Sales, however, were discontinued in 1999.
2.2. Branched DNAAssay
As a solid-phase sandwich assay based on bDNAtechnology (Fig. 1B), the Bayer
Versant (previously Chiron Quantiplex) assay involves the specific capture of HBV
genomic DNA to microwells by hybridization to complementary synthetic oligonu-
cleotide target probes (5,6). Detection of the captured HBV DNA is accomplished
through subsequent hybridization of bDNA amplifier molecules containing repeated
nucleotide sequences for the binding of numerous alkaline phosphatase-modified label
probes. Upon addition of a dioxetane substrate, the alkaline phosphatase-catalyzed light
emission is recorded as luminescent counts on a plate-reading luminometer. Light emis-
sion is proportional to the amount of HBVDNApresent in each specimen, and results
are expressed as milliequivalents per milliliter (Meq/mL).
The assay requires two 10-(cid:1)Laliquots of serum for each determination. Serum speci-
mens are measured in duplicate, and the quantity of HBVDNAis determined from a stan-
dard curve included on the same plate for each assay run. Four assay standards, prepared
by dilution of HBVDNA-positive human serum into HBVDNA-negative human serum,
which cover a 4 log range in concentration from approx 0.4 to 4000 HBVDNAmeq/mL,
10
are included. The assay standards are value-assigned against the primary HBVDNAstan-
dard representing the entire HBVgenome, subtype adw2, which is purified from recombi-
nant plasmid and quantified using different independent analytical methods (5,7).
2.3. DNA–RNAHybridization
This assay uses an HBV–RNAprobe to capture sample HBV DNAthat has been
rendered single-stranded (Fig. 1C). These hybrids are then bound onto a solid phase
with an anti-RNA–DNAhybrid antibody. This bound hybrid is reacted with antihybrid
antibody, which has been conjugated to alkaline phosphatase and reacts with a chemilu-
minescent substrate. The light emitted is measured on a luminometer, and the concen-
tration of HBVDNAis determined from a standard curve (8,9).
Overview of Commercial HBV Assay Systems 5
Recently, a second-generation (HBVDigene Hybrid Capture II) antibody capture
solution hybridization assay was developed (10). In this test, 30 (cid:1)L of serum sam-
ples, controls, and standards or calibrators are incubated with a denaturation reagent.
No additional sample preparation step is required. After preparation of the probe mix-
ture, an HBVRNAprobe is added to each well and incubated for 1 h. To capture the
DNA–RNAhybrids, an aliquot of the solution in the microplates is transferred to the
corresponding well of the anti-RNA–DNAhybrid antibody-coated capture microplate.
The hybrid is detected using an antihybrid antibody conjugated to alkaline phos-
phatase and detected with a chemiluminiscent substrate. To enable detection of HBV
DNAlevels of less than 1.42 × 105 copies/mL, the ultrasensitive format of the assay
is used. Here, 1-mL serum samples and controls along with 50 (cid:1)L of precipitation
buffer are centrifuged at 33,000g for 110 min at 4°C. The supernatant is discarded,
and the precipitated virus is dissolved. This procedure yields a 30-fold increase in
sensitivity(10).
2.4. Polymerase Chain Reaction
HBVDNAis isolated from 50 (cid:1)Lof serum by polyethylene glycol precipitation fol-
lowed by virion lysis and neutralization. Aknown amount of quantitation standard is
added into each specimen and is carried through the specimen preparation, amplifica-
tion, and detection steps subsequently used for quantification of HBVDNAin the spec-
imen(Fig. 1D).
In the Amplicor Monitor HBVtest a 104-bp segment of the highly conserved pre-
core–core region is amplified by PCR by using one biotinylated primer and one nonbi-
otinylated primer (11,12). The quantitation standard is amplified with the same primers
as target HBV. After 30 PCR cycles, HBV and quantitation standard are chemically
denatured to form single-stranded DNA. The biotinylated amplicon is then captured on
streptavidin-coated microwells and hybridized with HBVand internal standard-specific
dinitrophenyl (DNP)-labeled oligonucleotide probes. Following an incubation with
alkaline-phosphatase-conjugated anti-DNPantibodies and a colorimetric substrate, the
amount of HBVDNAin each specimen is calculated from the ratio of the optical den-
sity for the HBV-specific well to the optical density for the quantitation-standard-
specific well. The number of HBV DNA copies is calculated from a standard curve
prepared from each amplification run. If the result exceeds 4.0 × 107 HBV DNA
copies/mL, serum is diluted and retested.
The quantitative analysis of HBVDNAcan be automated using the Cobas Amplicor
Monitor HBVtest. In this system, viral DNAis still manually extracted. Quantitative
results of the Cobas Amplicor Monitor HBV test are interchangeable with measure-
ments by the manual microwell plate version of Amplicor (13). Future systems will also
automate extraction (e.g., Ampliprep), and fully automated analyzers will finally
become available.
2.5. Other HBVDNAQuantification Assays
Other HBV DNA quantification systems comprise the transcription-mediated
amplification (TMA)–based assay (14), the ligase-chain-reaction (LCR) assay (15),
6 Zeuzem
Fig. 1. Test principles of (A) Liquid hybridization assay (Genostics™ HBV-DNAAssay,
Abbott Laboratories), (B)Branched DNAassay (Versant HBV, Bayer Diagnostics), (C)Hybrid
Capture II Technology (Hybrid Capture™II System, Digene), and (D)Polymerase chain reaction
(Amplicor Monitor HBV, Roche Diagnostics).
the nucleic acid–based amplification (NASBA) assay (16), and various variations of
the mentioned technologies (17–22). TMA-, LCR-, and NASBA-based assays for
HBV DNAquantification are currently not commercially available in Europe or the
United States.
Overview of Commercial HBV Assay Systems 7
3. Sensitivity and Dynamic Range
Specimens tested with the liquid hybridization assay were considered positive for
HBVDNAat 1.5% of the positive control standard quantification value, or approx 1.6
pg/mL(3). The clinical quantification limit of the bDNAassay has been set at 0.7 HBV
8 Zeuzem
Fig. 2. Sensitivity and range of detection of different HBVDNAassays.
DNAmeq/mL(5). Similar to the HIV(Human Immunodeficiency Virus) or HCV(hep-
atitis C virus) RNAbDNAtests, sensitivity will be considerably improved in the next
version of the assay. The lower detection limit of the HBV DNA–RNAhybridization
capture assay in its ultrasensitive format is around 5000 copies/mL(10). The highest
sensitivity of HBVDNAquantification assays, however, is achieved by the PCR-based
assay (400 copies/mL) (13)(Fig. 2). Alimitation of this PCR assay is the relatively nar-
row linear range, requiring predilution of high-titer samples (13). These problems can
be solved by real-time PCR detection assays based on TaqMan technology (21–23). All
assay characteristics are summarized in Table 2.
4. Interassay Correlation Between HBVDNAQuantification Assays
The HBVDNAquantification values generated by the liquid hybridization assay are
expressed as pg/mL. Values of the branched DNAassay are expressed as MEq/mL, and
those of the DNA–RNAhybridization assay and the quantitative PCR are expressed as
copies/mL.
For evaluation of the theoretical relationship between pg and MEq/copies, the fol-
lowing assumptions are required (24):
• HBVDNAcomprises 3200 base pairs
• The molecular weight of a base pair is 666 g/mole
• Avogadro’s number = 6.023 ×1023molecules or copies mole.
According to the following calculations:
• 3200 base pairs ×666 g/mole = 2.13 ×106g/mole
• (6.023 ×1023copies/mole)÷(2.13 ×106g/mole) = 2.83 ×1017copies/g
Overview of Commercial HBV Assay Systems 9
Table 2
Comparison of the Characteristics of Different HBVDNAQuantification Assays
Liquid Branched DNA DNA-RNA Polymerase
hybridization assay hybridization chain
assay (Bayer Diag.) assay reaction assay
(Abbott Lab.) (Digene II) (Roche Molec.
Systems)
Volume 100 (cid:1)L 2 ×10(cid:1)L 30 (cid:1)L/1 mL 50 (cid:1)L
Sensitivity
pg/mL 1.6 2.1 0.5 / 0.02 0.001
copies/mL 4.5 ×105 7×105 1.4×105/ 5 ×103 4×102
Linearity 5 ×105–approx 1010 7×105–5×109 2×105–1×109 4×102–1×107
(copies/mL) 5 ×103–3×106 Cobas: 106
TaqMan: 1010
Genotype D>A A,B,C,D,E,F A,B,C,D (A),B,C,D,E
independence
Coefficient of 12–22% 6–15% 10–15% 14–44%
variation
• (2.83 ×1017copies/g) ÷(1 ×1012g/pg) = 2.83 ×105copies/pg
The theoretical conversion equation is calculated as 1 pg/mL= 2.83 ×105copies/mL
= 0.283 meq/mL.
Several direct comparisons among different assays have been performed
(8,9,23–31). Conversion factors are summarized in Fig. 3. Large discrepancies were
observed between the liquid hybridization assay and the other signal and target amplifi-
cation systems. Agood concordance exists between the DNA–RNAhybridization assay
(Hybrid Capture II) System and the quantitative PCR detection assay (Amplicor Moni-
tor HBV).
5. Standardization of HBVDNAAssays
Different extraction procedures of HBVDNAfrom serum generate different results
in hybridization assays when compared with cloned DNA(32). Because HBVcontains
viral polymerase covalently bound to genomic DNA, extraction procedures that remove
protein from DNAextract the HBVDNAtogether with the polymerase. Proteinase K
digestions of serum or plasma are often incomplete, and, thus, losses of HBV DNA
occur during the subsequent phenol extraction. In contrast, lysis procedures without
proteases do not remove a large amount of plasma protein, which may interfere with the
assay. Cloned HBVDNAwithout covalently bound polymerase binds less efficiently to
filters than does the virion-derived HBVpolymerase/DNAcomplex in the presence of
large amounts of plasma proteins. Thus, cloned HBVDNAcannot directly be used as a
reference sample for virion-derived HBVDNAunless the polymerase and plasma pro-
tein have been carefully removed from the sample. Purity and quantity of cloned HBV
DNAhave to be assessed accurately.
10 Zeuzem
Fig. 3. Correlation between HBVDNAassays. Concentration ranges (< 30; 30–500; > 500)
are given in pg/mL.
In view of these problems, the Eurohep Pathobiology Group decided to generate two
reference plasma samples for HBV DNA. Plasma donations from two single, highly
viremic carriers of HBVgenotype A(HBVsurface antigen subtype adw2) and genotype
D (awy2/3), respectively, were collected, and the accurate number of HBVDNAmole-
cules was determined (2.7 ×109and 2.6 ×109HBVDNAmolecules/mL, respectively)
(33). Genotypes A and D are predominant in Europe and North America. Pooling of
donations from different HBVcarriers was avoided because many infected patients carry
antibodies against epitopes of heterologous HBVgenotypes. This could cause aggrega-
tion of HBVand difficulties in testing of dilutions made from the reference samples. The
two Eurohep reference plasma samples have already been used for the standardization of
test kits (25)and in quality control trials (34), and the plasma from the carrier of geno-
type Awill be the basis of a World Health Organization (WHO) reference sample.
6. Clinical Impact of HBVDNAQuantification
Quantitative detection of HBV DNA allows identification of patients with highly
replicative hepatitis B who are HBeAg-negative (35). Furthermore, HBVDNAquan-
tification in serum or plasma provides a means of measuring the viral load in patients
before, during, and after antiviral therapy. There appears to be a level of HBV DNA
below which hepatitis B is inactive and nonprogressive; this level may vary within the
patient population and depending on the assay may be as high as 106 to as low as 104
copies mL(1,35). Nevertheless, cases with suppressed HBVactivity, despite the very
low levels of viremia, maintain a relatively high amount of intrahepatic viral genomes
(36). The generation of treatment-resistant HBVmutants can be suspected when serum
HBVDNAincreases in patients during therapy. Furthermore, the level of HBVDNA
makes it possible to estimate the potential infectivity of HBV-infected patients. Highly
Overview of Commercial HBV Assay Systems 11
sensitive tests for HBVDNAare useful for detection of blood donors who express no
serological markers and for detection of HBV in therapeutic plasma protein prepara-
tions(37).
7. Conclusions
HBV DNA quantification assays suffer limitations in standardization. The liquid
hybridization assay produced HBV DNA levels that are 10- to 80-fold lower than
results reported from the bDNAassay and 10–20 times lower than the Digene Hybrid
Capture assay. Different assays also have different linear ranges of accuracy. The intro-
duction of the WHO HBVDNAstandard will facilitate standardized quantification. In
the future, a panel of standards for all HBV genotypes may be necessary to achieve
genotype-independent HBVDNAquantification.
In view of the limitations surrounding viral assays, it is currently still difficult to
assess the clinical significance of different levels of HBVDNA. Empirally, it appears
that patients with an inactive carrier state generally have viral load of less than 105–106
copies/mL, whereas patients with an active carrier state exhibit HBVDNAlevels above
105–106 copies/mL. High-sensitivity quantification of HBV DNAmay particularly be
clinically useful in the diagnosis of HBeAg-negative patients and for monitoring
response to therapy. Careful assessment of the clinical implications of different viral
levels using standardized reagents is much needed. In addition to HBVDNAquantifi-
cation, clinical evaluation of HBV genotyping assays and molecular tests for specific
mutations (pre-core, core promotor, surface, and polymerase) are required (38).
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