Journal of Viral Hepatitis, 2003, 10, 331–334
A rapid immunochromatographic assay for hepatitis B
virus screening
D. T.-Y. Lau,1 H. Ma,1 S. M. Lemon,2 E. Doo,3 M. G. Ghany,3 E. Miskovsky,1 G. L. Woods,4
Y. Park,3 and J. H. Hoofnagle,3 Departments of 1Internal Medicine and 2Microbiology & Immunology, The University of Texas Medical
Branch at Galveston, Galveston, TX; 3Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD; and 4Department of Pathology, The University of Texas Medical Branch at Galveston, Galveston, TX, USA
Received September 2002; accepted for publication November 2002
SUMMARY. Simple, rapid and accurate assays for hepatitis B
surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are
helpful for clinical diagnosis and field epidemiological surveys. A
commercially developed, rapid immunochroma-tographic test for
simultaneous detection of HBsAg and HBeAg was evaluated using
a total of 2463 selected samples (827 frozen sera, 1011 fresh sera,
and 625 whole blood samples). Results of the rapid test were
compared with standard enzyme immunoassay (EIA) methods for
HBsAg and HBeAg detection. The accuracy of the rapid test was
excellent and was similar for frozen sera, fresh sera and whole
blood. The overall sensitivity and specificity for the detection of
HBsAg were 95 and 100%, and the corres-ponding positive and
negative predictive values were 100
INTRODUCTION
Hepatitis B virus (HBV) infection is a major public health
problem, responsible for considerable morbidity and mor-tality
from chronic liver diseases [1]. Chronic hepatitis B affects over
300 million people worldwide and is especially prevalent in
developing countries in Asia and Africa. Even though hepatitis
B is less common in North America, it is estimated to affect at
least one million Americans [2]. There has been significant
progress in the diagnosis, treatment and prevention of hepatitis
B in the last few decades. However, access to health care and
standard laboratory technology for management of hepatitis B
remain significant problems in
Abbreviations: EIA, enzyme immunoassay; HBeAg, hepatitis B e
antigen; HBsAg, hepatitis B surface antigen; ICT, immunochro-
matographic test.
Correspondence: Daryl T.-Y. Lau, Assistant Professor of Medicine,
Division of Gastroenterology and Hepatology, Department of Inter-nal
Medicine, University of Texas Medical Branch at Galveston
4.106 McCullough Building, 301 University Boulevard, Galveston,
TX 77555-0764, USA. Tel.: 409-772-1501; Fax: 409-772-1343; E-
mail: dalau@utmb.edu
2003 Blackwell Publishing Ltd
and 99.7%, respectively. The sensitivity and specificity for the
detection of HBeAg were slightly less than that for HBsAg,
and were 80 and 98%, with positive and negative predictive
values of 91 and 94%, respectively. Thus, compared with the
EIA method, the rapid test was highly sensitive and accurate
for the detection of HBsAg although somewhat less sensitive
and specific for detection of HBeAg. Because of its speed,
simplicity and flexibility, the rapid test is ideally suited for
HBsAg and HBeAg screening in population-based epidemio-
logical studies and in low risk populations, particularly in
regions of the world where hepatitis B is endemic.
Keywords: diagnosis, enzyme immunoassays, hepatitis B,
immunochromatographic assay.
many developing countries, and in some rural as well as urban
areas of North America. An accurate, simple and inexpensive
diagnostic test would be valuable for population-based HBV
screening programmes, especially in countries where hepatitis
B is endemic. The aim of this study was to compare the
sensitivity and specificity of a recently developed rapid
immunochromatographic test (ICT) to that of enzyme
immunoassays (EIAs) that have been approved by the U.S.
Food and Drug Administration (FDA) for the detection of
hepatitis B surface antigen (HBsAg) and hepatitis B e antigen
(HBeAg). The accuracy of the rapid test using frozen stored
sera, fresh sera and whole blood was also determined.
MATERIALS AND METHODS
Blood samples
A total of 2463 human samples from four sources were
available for the study: (1) frozen sera (n ¼ 827): samples
were collected from incarcerated offenders on entry to the
Texas Department of Criminal Justice (TDCJ) system. These
samples had been stored frozen at )70 LC for approximately 1
year before the study. (2) Fresh sera (n ¼ 1011): samples
332 D. T.-Y. Lau et al.
were collected from patients in hepatology clinics at the
University of Texas Medical Branch at Galveston; the rapid
ICT test was performed on the same day of blood collection
with these samples. (3) Fresh whole blood (n ¼ 625): sam-ples
were obtained from participants in a Chinese Commu-nity
Health Fair in Houston, TX and from patients with known
hepatitis B infection at the Liver Diseases Clinic at the Clinical
Center of the National Institutes of Health, Bethesda, MD.
Fresh whole blood samples were tested by the rapid test within
a few minutes of blood collection. Serum was then separated
from the whole blood and later tested for HBsAg and HBeAg
using standard EIA methods. The results of the rapid test
results were interpreted without the knowledge of clinical data
or EIA results.
HBV assays
Standard, FDA-approved EIA assays for HBsAg (ETI-MAK-2)
and HBeAg (ETI-EBK) (DiaSorin Inc., Stillwater, MN, USA)
were carried out according to the manufacturer’s instruction
manuals. HBsAg assays were performed on all 2463 sam-ples,
while HBeAg assays were performed only on samples that
were HBsAg positive by EIA or the rapid test.
The rapid hepatitis B assay is an in vitro qualitative
immunochromatographic test (ICT) that can detect both
HBsAg and HBeAg simultaneously. It was first developed by
AMRAD ICT (French Forest, NSW, Australia) in Australia,
but in June, 2000 was acquired by Binax Inc., (Portland Maine,
USA) which now manufactures the ICT Hepatitis B sAg/eAg
test under the Binax NOW label in the United States. The test
utilizes two sets of antibodies specific to HBsAg and HBeAg
and captures the antigens in a standard sandwich format. The
first set of antibodies is labelled with colloidal gold and is
impregnated in a test pad. The second set is immobilized as two
separate lines on a cellulose membrane that oppose the test pad
in a bi-folded card. When whole blood (100 lL) or serum
sample (100 lL) is added to the test pad, HBsAg and HBeAg
present in the sample bind to the colloidal gold-labelled
antibodies. The card is then closed and the serum diffuses from
the test pad into the membrane. The HBsAg and HBeAg
antibody complexes are captured by their respective antibodies
immobilized on the membrane. The captured antigen–antibody
complexes are visualized by the colloidal gold yielding a pink
colour reaction that is visible to the unaided eye. The reaction
is usually visible within 2 min, but low titre samples may
require up to 15 min to develop. For the HBV negative
samples, no anti-body complex is captured on the membrane
and the anti-bodies diffuse through the membrane and are not
concentrated at the site of the immobilized antibodies. The
components of the rapid test and an example of positive
reaction are shown in Fig. 1.
All the frozen and fresh sera included in this study were
tested using the AMRAD ICT manufactured test. Among the
625 whole blood samples, 464 (74%) were tested using the
2003 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 10, 331–334
AMRAD ICT manufactured test and 161 (26%) using the
Binax manufactured test. As the two rapid tests yielded 100%
concordant results on 60 random samples, the results of the
625 whole blood samples were combined for analysis.
RESULTS
Results of the standard EIA assay and the rapid ICT test for
detection of HBsAg and HBeAg on frozen sera, fresh sera and
whole blood samples are summarized in Tables 1 and 2. For
frozen stored sera, the rapid test correctly identified 55 of the
58 HBsAg-positive samples and all HBsAg-negative samples
by EIA. Thus, the rapid test had good sensitivity (95%) and
specificity (100%) for the detection of HBsAg. The rapid test
correctly identified 16 of the 20 HBeAg-pos-itive samples, and
misdiagnosed two of 38 negative sera. Its sensitivity and
specificity for the detection of HBeAg were, therefore, 80 and
95%, respectively. For fresh sera, the rapid test was 94%
sensitive and 100% specific for the detection of HBsAg and
80% sensitive and 100% specific for the detec-tion of HBeAg.
For whole blood samples, the results of the rapid tests were
similar. The ICT test correctly identified 72 of 75 HBsAg-
positive and all HBsAg-negative samples, yielding 96%
sensitivity and 100% specificity. The rapid ICT test correctly
identified 12 of 15 HBeAg-positive and 60 of 61 HBeAg-
negative samples. The sensitivity and specificity for HBeAg in
whole blood were, therefore, 80 and 98%, respectively.
As the results of the rapid ICT test were similar for frozen
sera, fresh sera and whole blood, all the results from the three
groups were pooled for combined analysis. The rapid test
correctly identified 139 of 146 HBsAg-positive and all 2298
HBsAg-negative samples. The sensitivity and specific-ity for
the detection of HBsAg in this entire cohort were thus 95 and
100% and the corresponding positive and negative predictive
values for HBsAg were 100 and 99.7%, respect-ively. The
rapid test was able to identify 32 of 40 HBeAg-positive, and
misdiagnosed three of 124 negative samples. Its sensitivity and
specificity for the detection of HBeAg were 80 and 98%, and
its positive and negative predictive values were 91 and 94%
respectively.
The samples with false negative results by the rapid test
generally had much lower optical density (OD) readings by
EIA. For HBsAg, the average EIA OD values were 0.489 for
the rapid test negative samples compared with 2.395 for the
positive samples by rapid test. Similarly for HBeAg, the
average EIA OD readings were 0.584 for the rapid test
negative samples and 2.561 for the rapid test positive samples.
DISCUSSION
The results of this study indicate that the ICT Hepatitis B
sAg/eAg rapid test is highly sensitive and accurate in the
detection of HBsAg. The rapid test also had excellent
 Rapid HBV immunochromatographic assay 333

Fig. 1 The components of the rapid test

and an example of a sample that tested
positive for both HBeAg and HBsAg.

Table 1 Comparison of ICT rapid test with

Frozen sera Fresh sera Whole blood
standard EIA assay for detection of
HBsAg and HBeAg + ) n + ) n + ) n

HBsAg rapid test + 55 0 55 30 0 30 72 0 72

) 3 769 772 2 979 981 3 550 553
Total 58 769 827 32 979 1011 75 550 625
HBeAg rapid test + 16 2 18 4 0 4 12 1 13
) 4 36 40 1 25 26 3 60 63
Total 20 38 58 5 25 30 15 61 76

specificity for the detection of HBeAg, but was less sensitive
than the standard EIA method. Although EIAs are consid-ered
to be the gold standard for detection of HBsAg and HBeAg, the
rapid test has several advantages. The rapid ICT test is simple
and requires no specific laboratory instru-ments, reagents, or
storage conditions. It can be carried out
using small blood samples that can easily be obtained by finger
pricks. The ICT reagents can be stored for as long as 3 months
at room temperature (15–30 LC). The rapid test can be
performed by personnel with minimal training in laboratory
techniques. Generally, the results are available within 5 min.

2003 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 10, 331–334

334 D. T.-Y. Lau et al.
HBsAg HBeAg
Frozen Fresh Whole Frozen
sera sera blood All sera
Sensitivity 94.5 93.8 96.0 95% 80.0
Specificity 100 100 100 100% 94.7
Several other rapid diagnostic methods for detection of
HBsAg have been developed and evaluated [3–5]. The rapid
test used in this study was an immunochromatographic test.
Compared with the other rapid tests, such as the reverse-
passive haemagglutination and latex agglutination assays, the
ICT-based assay appears to be superior in speed, flexibility and
simplicity [6–9]. For some ICT-based assays, only serum or
plasma can be used because red blood cells interfere with the
reading of test results. However, the currently manufactured
ICT rapid test assay employs a new method that was developed
for use with whole blood in ICT-based assays [10]. To date, the
ICT Hepatitis B sAg/eAg test is the only assay capable of
detecting HBsAg in whole blood, serum or plasma in a single
step without pretreatment of blood. Our results confirm that the
accuracy of the rapid test is similar for frozen stored sera, fresh
sera or whole blood.
The sensitivity of the rapid test evaluated in this study was
similar to other rapid tests for the detection of HBsAg. A
significant distinction of the ICT rapid test is its ability to
detect both HBsAg and HBeAg simultaneously. Other rapid
testing methods can detect only HBsAg, or only anti-HBs in
separate assays [10]. HBeAg can be important in the eval-
uation of patients with chronic hepatitis B and its presence
generally correlates with higher levels of viraemia and
infectivity and worse prognosis [11,12]. Because of its
accuracy and simplicity, the ICT rapid test is ideal for pop-
ulation-based HBV screening programmes to determine the
HBV carrier rates. For patients who test HBsAg-positive by the
ICT Hepatitis B sAg/eAg assay but negative for HBeAg, it may
be useful to consider further evaluation using a standard EIA
test to confirm the presence or absence of HBeAg, as the EIA
is more sensitive for the detection of low titre HBeAg.
In conclusion, the ICT Hepatitis B sAg/eAg rapid test is an
accurate, simple and inexpensive diagnostic test that may have
advantages over standard assays for population-based HBV
surveillance programmes, field epidemiology and rural clinics
that provide HBV treatment, especially in countries where
hepatitis B is endemic.
ACKNOWLEDGEMENTS
Binax and Glaxo provided the rapid test reagents but no other
financial support for this study.
2003 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 10, 331–334
Table 2 Sensitivity and specificity of the
ICT rapid test for HBsAg and HBeAg
Fresh Whole
sera blood All
80.0 80.0 80%
100 98.4 98%
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