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www.elsevier.com/locate/jim
Journal of Immunological Met
Research paper
Development of a rapid, immunochromatographic strip test for serum
asialo a1-acid glycoprotein in patients with hepatic disease
Eun Young Lee a,1, Ji Hyun Kang a, Kyoung A. Kim a, Tai Wha Chung b, Hee Jung Kim c,
Do Young Yoon a, Hee Gu Lee a, Dur Han Kwon a, Jae Wha Kim a,
Cheorl Ho Kim d, Eun Young Song a,*
a The Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong,
Daejeon 305-600, Republic of Koreab Neobiodigm, Venture Bldg., Bio21 Foundation Center, Jinju-City 305-6, Republic of Korea
c Catholic University, St. Mary’s Hospital, Daejeon 301-723, Republic of Koread Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju 780-714, Republic of Korea
Received 13 July 2005; received in revised form 12 October 2005; accepted 20 October 2005
Available online 12 December 2005
Abstract
Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with
hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo a1-
acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a
diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step
immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal
antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which
generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with
colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to
form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the
immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample
specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with
the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP
continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (N1.5 Ag/mL) yielded a result line
and a control line, whereas an AsAGP-negative specimen (b1.5 Ag/mL) produced only a single control line. The entire test
procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the
immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by
ELISA. The two methods showed excellent correlation, with 83–100% above/below the cut-off value (1.5 Ag/mL). Therefore,
0022-1759/$ - s
doi:10.1016/j.jim
Abbreviation
hepatocellular c
pH 7.4; PAGE,
* Correspondin
E-mail addre1 Present addr
hods 308 (2006) 116–123
ee front matter D 2005 Elsevier B.V. All rights reserved.
.2005.10.010
s: AGP, a1-acid glycoprotein; AsAGP, asialo a1-acid glycoprotein; mAb, monoclonal antibody; LC, liver cirrhosis; HCC,
arcinoma; RCA, ricinus communis agglutinin; HG, haptoglobin; MG, a2-macroglobulin; PBS, 0.1M phosphate, 0.15M NaCl,
polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.
g author. Tel.: +82 42 860 4138; fax: +82 42 860 4593.
ss: [email protected] (E.Y. Song).
ess: Food and Drug Division, Ulsan Institute of Health and Environment, Yaum, Ulasan 576-10, Korea.
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E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123 117
we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich
ELISA.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Asialo a1-acid glycoprotein; Immunochromatographic strip; Monoclonal antibody; Hepatic disease; Serum marker
1. Introduction
Circulating plasma glycoproteins are synthesized,
metabolized, and cleared by the liver. According to
the mechanism, terminal sialic acid residues of the
glycoproteins are hydrolyzed to expose penultimate
galactose residues, and the asialoglycoprotein receptors
of hepatic cells specifically capture and catabolize the
desialylated glycoproteins (Ashwell and Morell, 1974;
Neufeld and Ashwell, 1979; Well et al., 1980; Bridges
et. al., 1982; Weigel and Oka, 1982; Baenziger, 1984).
It has been reported that the circulating desialylated
glycoprotein (asialoglycoprotein) levelswere significantly
elevated in patients with hepatic disease as compared with
those in normal individuals (Sawamura and Shiozaki.,
1991). Additional studies showed a correlation between
the serum asialoglycoprotein levels and the clinical sta-
tus or functionality of the liver in patients with hepatic
disease (Marshall et al., 1974; Sawamura and Shiozaki,
1991). Thus, serum asialoglycoprotein level was sug-
gested as an indicator for assessment of the clinical
statuses of some hepatic diseases, such as hepatitis,
liver cirrhosis, and hepatocellular carcinoma (Marshall
et al., 1974; Reffetoff et al., 1975; Arima and Sci., 1979;
Serbource-Goguel et al., 1983; Sawamura et al., 1985).
Several methods have been developed to quantita-
tively measure serum asialoglycoprotein levels (Van
and Ashwell, 1972; Reffetoff et al., 1975; Arima and
Sci, 1979; Serbource-Goguel et al., 1983; Sawamura et
al., 1985). Recently, a solid-phase sandwich assay was
developed to detect one of the asialoglycoproteins,
asialo-a1-acid glycoprotein (AsAGP), in serum (Song
et al., 2003). The assay system was composed of
monoclonal antibody (mAb) to a1-acid glycoprotein
(AGP) as capture protein and an enzyme-conjugated
lectin, which recognizes asialo a1-acid glycoprotein, as
probe protein. The results of clinical study revealed an
elevation of serum AsAGP level primarily in patients
with LC or HCC. Thus, serum AsAGP was suggested
as a useful differential marker for LC or HCC and
warrants further investigation (Song et al., 2003).
In this study, we have developed a rapid, one-step
immunochromatographic strip which is capable of spe-
cifically detecting AsAGP in serum specimen. Although
ELISA can measure the concentration of serum AsAGP
and be used for diagnosing hepatic disease, it takes about
3h to complete the measurement and the assay requires a
special instrument. Newly developed immunochromato-
graphic strip can be overcome these shortcomings, rapid
and convenient. Therefore, it could be applied to diag-
nosis of hepatic disease at home or in hospital.
2. Materials and methods
2.1. Samples
Serum specimens were obtained from the Catholic
University, St. Mary’s Hospital (Daejeon, Korea). Con-
sent from each patient was obtained for research use of
the serum specimens, and the protocols were in accor-
dance with the guidelines approved by the committee at
the Catholic University, St. Mary’s Hospital (Daejeon,
Korea).
2.2. Materials
a1-Acid glycoprotein (AGP), haptoglobin (HG), and
a2-macroglobulin (MG) were obtained from Sigma (St.
Louis, MO, USA). The purified lectin, ricinus communis
agglutinin (RCA), was purchased fromE.Y. Laboratories
(San Mateo, CA, USA). Desialylation of AGP was car-
ried out in 0.1NH2SO4, followed by neutralization with
1N NaOH and dialysis against PBS buffer. The desialy-
lated AGP was further purified by Sephadex G-200
column chromatography. For the immunochromato-
graphic strip, colloidal gold microparticles were pur-
chased from BBI international Ltd (Cardiff, CF14
5DX, UK), membranes were obtained from Schleicher
and Schell GmbH (Dassel, Germany) and Whatman
(Fairfield, NJ, USA). The dispensing platform and cut-
ting module were obtained from Bio-Dot Inc. (Irvine,
CA, USA). The scanning densitometer, CS-9300PC,
was purchased from Shimadzu (Kyoto, Japan).
2.3. Production of monoclonal antibodies (mAbs)
2.3.1. Preparation of asialo-a1-acid glycoprotein
(AsAGP)
Desialylated AGP was prepared in the following
manner: 60 mg of the AGP was dissolved in 12 mL
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AsAGPRCA
Gal
Colloidal Gold Microparticles
Anti-AGP antibody
ig. 1. A schematic representation of microparticle-based immunoas-
ay. Microparticle-based immunoassay in which monoclonal antibody
Ab) to a1-acid glycoprotein (AGP) and galactose-binding lectin,
cinus communis (RCA), have been employed as capture protein and
robe protein, respectively. Serum AsAGP binds mAb to AGP onto
e colloidal gold microparticles and then bind lectin (RCA) onto the
embrane.
E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123118
of 0.1N H2SO4 and was hydrolyzed. The mixture was
then neutralized with 1N NaOH and dialyzed against
0.01M phosphate buffer, pH 7.4. The precipitate
formed during hydrolysis was removed by centrifuga-
tion, and the supernatant solution was applied to a
Sephadex G-200 column. The protein was eluted and
dialyzed against the buffer. The protein concentration
was determined using the Bradford method (Bio-Rad
Labs, Hercules, CA, USA).
2.3.2. Production of monoclonal antibodies
The procedure employed for the production of
monoclonal antibodies was based on the protocol of
Kohler and Milstein (1975). Balb/c mice were immu-
nized by intraperitoneal injection of AsAGP mixed with
TiterMax (CytRx Corporation, Georgia). The spleen
cells of the mice were fused with the SP2/0-Ag14
murine myeloma cell line. The mAb-producing hybri-
domas were cloned by the limiting dilution technique,
and the selected hybridoma clones were propagated by
injection into mice. In order to obtain purified mAb, the
ascitic fluids were subsequently harvested and pro-
cessed by Protein G–Sepharose 4B chromatography.
AGP-specific mAb was selected based on its ability
to bind AsAGP.
2.3.3. Western blot analysis
Sodium dodecyl sulfate–polyacrylamide gel electro-
phoresis (SDS-PAGE) experiment was performed using
the Past PAGE System of Pharmacia (Piscataway, NJ,
USA) according to the instructions of the manufacturer,
using 10% gel. Gel was transferred, probed with mAb
to AGP and then with anti-mouse IgG-HRP. The gel
was subsequently developed using dimethylaminoben-
zidine solution.
2.4. Development of immunochromatographic strip
The immunochromatographic strip for AsAGP
assay is a microparticle-based immunoassay. mAb to
AGP and galactose-binding lectin, RCA were em-
ployed as capture protein onto microparticles and
probe protein onto nitrocellulose membrane, respec-
tively (Fig. 1). Immunochromatographic strip consisted
of a sample pad, a conjugate pad, a nitrocellulose
membrane, and an absorption pad (Fig. 2). The conju-
gate pad contained colloidal gold microparticles-con-
jugated mAb to AGP. The nitrocellulose membrane
was used as a chromatographic support on which the
RCA and anti-mouse IgG were immobilized. The cel-
lulose membrane was used as a sample pad and an
absorption pad.
F
s
(m
ri
p
th
m
2.4.1. Preparation of colloidal gold microparticle-
conjugated mAb As16.89
The mAb As16.89 was conjugated with colloidal
gold microparticles by the following method. In brief,
15 Ag of the purified mAb As16.89 in 2 mM borax was
added to 500 AL of colloidal gold microparticles (40
nm diameter), and incubated for 3 h in order to conju-
gate the immunoglobulin with the colloidal gold micro-
particles. After centrifugation at 12,000 rpm for 40 min
at RT, the colloidal gold microparticles-conjugated
mAb As16.89 were collected in the pellet form. The
supernatant was removed, and the pellet was suspended
in 2 mM borate buffer. The colloidal gold microparti-
cles-conjugated mAb As16.89 were washed three times
with borate buffer in the same manner as described
above. Finally, the pellet was resuspended in 2mM
borate buffer containing 1% BSA. The colloidal gold
microparticles-conjugated mAb As16.89 suspension
was dispensed onto the conjugate pad.
2.4.2. Immobilization of lectin onto nitrocellulose
membranes
Using a dispenser, 2.0 mg/mL of RCA was immo-
bilized in a straight line along a width of 300 mm, at a
distance of 0.8 cm from the edge of a nitrocellulose
membrane. This was used as a result line to bind the
AsAGP/colloidal gold microparticles-conjugated mAb
As16.89 complexes. The membrane was dried at 37 8C1 mg/mL of anti-mouse IgG was also immobilized on
the nitrocellulose membrane in a straight line along a
width of 300 mm, at a distance of 0.8 mm from the
result line. This was used as a control line to confirm
the success of the test.
2.4.3. Assembly of the immunochromatographic strip
The conjugate pad containing colloidal gold
microparticles-conjugated mAb As16.89 was cut
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A.
Sample padConjugate pad Result line Control line Absorption pad
B.
Plastic backing for adhesiveAbsorption padConjugate pad
Membrane
Sample pad
Fig. 2. Schematic diagram of the immunochromatographic strip for AsAGP. (A) Top view. (B) Side view. The strip consists of a conjugate pad, a
nitrocellulose membrane, a cellulose membrane, and a covering case. The conjugate pad contains mAb against AsAGP conjugated with a colloidal
gold microparticle. In the detection zone, the nitrocellulose membrane is used as a chromatographic support on which the RCA and rabbit anti-
mouse IgG are immobilized. The cellulose membrane is used as sample pad and absorption pad.
E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123 119
into 5-mm-long and 5-mm-wide strips. The RCA and
anti-mouse IgG immobilized nitrocellulose membrane
was then sliced into 25-mm-long and 5-mm-wide
strips. A conjugate pad was attached to one side of
a nitrocellulose membrane and was covered with
sample pad. A cellulose membrane (absorption pad)
for absorption excess serum and AsAGP/colloidal
gold microparticles-conjugated mAb As16.89 was
then attached to the other side of the nitrocellulose
membrane strip.
2.4.4. AsAGP determination in serum using the
immunochromatographic strip
Ten-fold diluted serum sample (100 AL ) was ap-
plied onto sample pad. The serum sample was passed
over the conjugate pad and moved into the nitrocellu-
lose membrane. The visible color was appeared at
control line and/ or result line within 5 min. AsAGP
positive specimen yielded two lines at a result line and
a control line, whereas an AsAGP negative specimen
produced only one control line.
2.5. mAb-lectin sandwich ELISA for AsAGP
A 96-well microtiter plate was coated overnight
with 100 AL per well of 1.0Ag/mL mAb to AGP at
4 8C and washed with PBS solution. The wells
were then overcoated with 3% BSA solution. Ten-
fold diluted serum samples were applied in duplicate
at 100 AL per well and were allowed to react with
the mAb for 2 h at room temperature. After the
wells were washed with PBS solution, appropriately
diluted HRP-conjugated lectin, RCA, was applied at
100 AL per well for 1 h. Orthophenylenediamine
(OPD) solution was subsequently added to the wells
and, after the reaction was stopped with 1N H2SO4
solution, the absorbance was measured at 490 nm
on an ELISA reader (Molecular Device, Sunnyve,
CA, USA).
3. Results
3.1. Production and characterization of antibodies to
AsAGP
To obtain monoclonal antibody, the purified
AsAGP and AGP were immunized to mice, respec-
tively. Among the hybridomas generated against AGP
and AsAGP, four clones produced mAbs that strongly
bound AsAGP and AGP. The immunoglobulin iso-
typing showed that three clones (As1.71, As4.13,
and As1.1) were IgM class; one clone (As16.89)
was IgG2b (small) class. mAb As16.89 was specifi-
cally selected for this investigation because of its high
affinity to AGP and AsAGP. The specificity of mAb
As16.89 was determined by the Western blot analysis
and ELISA. mAb As16.89 was bound to both AsAGP
and AGP, even though it was prepared after immuniz-
ing AsAGP and AGP, respectively. However, there
was no cross reaction with haptoglobin or a2-macro-
globulin (Fig. 3).
3.2. Development of immunochromatographic strip for
AsAGP
The serum sample was applied onto sample pad
and passed over the conjugate pad and moved into
nitrocellulose membrane as a result of capillary mi-
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Control line
Result line
Positive Negative
ig. 4. Immunochromatographic strip test for AsAGP. Serum speci-
en was applied to the sample pad. AsAGP positive specimen
1.5 Ag/mL) yielded a result line and a control line, whereas
sAGP negative specimen (b1.5 Ag/mL) produced only one result
ne. The entire test procedure was completed in less than 5 min.
E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123120
gration. AsAGP in the serum subsequently was
bound to the colloidal gold microparticles-conjugated
mAb As16.89. Finally, the AsAGP/colloidal gold
microparticles-conjugated As16.89 complex was
trapped by the immobilized RCA on the result line
and anti-mouse IgG on the control line. Detection of
AsAGP with this immunochromatographic strip was
completed within 5 min.
To investigate the detection limit of the immuno-
chromatographic strip, 10 Ag/mL of AsAGP was
spiked in normal serum sample and serially diluted.
A serum sample containing AsAGP at less than 1.5
Ag/mL appeared in visible color on the control line,
but did not appeared a color on the result line.
Serum control samples containing AsAGP at above
1.5 Ag/mL, on the other hand, appeared in visible
color on the result line as well as the control line
(Fig. 4). These results suggested that the detection
limitation of AsAGP in serum was 1.5 Ag/mL with
the naked eye.
In order to examine the dose–response phenome-
non (the color intensity in the result line) of the
newly developed immunochromatographic strip test,
AsAGP was spiked in the normal samples (4.0 Ag/mL, 3.0 Ag/mL, 2.0 Ag/mL, and 1.5 Ag/mL, respec-
tively). The color intensity in the result line increased
in a dose–response manner of AsAGP (Fig. 5). This
differential color intensity represented the concentra-
tion of AsAGP and would be applicable as a sensor
for quantification of results.
A B
0.5
1
1.5
2
2.5
0 1 2 3 4 5 6 7 8 9
O.D
.
AGPAsAGPHaptoglobinα2-macroglobulin
Dilution factor (10-n )
4 51 2 3
Fig. 3. Characterization and specificity of mAb against AsAGP
(As16.89) by ELISA and Western blot analysis. (A) ELISA. A
microtiter plate was coated with mAb (As16.89) and overcoated
with 1% BSA. One hundred AL of serially diluted AsAGP, AGP,
a2-macroglobulin, and haptoglobin was added per well and incu-
bated for 2 h at room temperature. Following the incubation, a 500-
fold dilution of HRP-conjugated RCA was reacted for l h, and the
color development was measured by the spectrophotometer at 492
nm. (B) Western blot analysis. Lane 1, MW markers; Lane 2, AGP;
Lane 3, AsAGP; Lane 4, a2-macroglobulin; Lane 5, haptoglobin.
F
m
(N
A
li
The cross-reactivity of the immunochromatographic
strip for AsAGP was examined. The presence of AGP
(up to 10.0 Ag/mL), haptoglobin (up to 10 Ag/mL), and
a2-macroglobulin (up to 10.0 Ag/mL) did not interfere
with the results of the immunochromatographic strip
test (Fig. 5).
3.3. Comparison of the immunochromatographic strip
test with the sandwich ELISA
In order to examine the reliability of testing proce-
dures, we carried out the immunochromatographic
strip test with 102 serum samples and compared the
results of these tests with the results obtained by the
ELISA. The table was summarized the comparison
between the two methods (Table 1). As described,
the two methods were shown to be in excellent agree-
ment in diagnostic judgment. The agreement rate
based on the data in Table 1 was 83–100% above/
below the cut-off value (1.5 Ag/mL). We therefore
concluded that the results of the immunochromato-
graphic test are in excellent accordance with the
results of the sandwich ELISA.
3.4. Determination of serum AsAGP by
immunochromatographic strip
AsAGP presence/absence in serum was determined,
after 10-fold dilution, by immunochromatographic
strip. The results were summarized based on the dis-
ease category (Table 2). About 90% of patients with
LC or HCC, as well as those who had both LC and
HCC, were shown positive signal at the result line and
97% of normal subjects were shown negative signal.
However, about 40–50% of patients with hepatitis
(acute, chronic) was not detected as AsAGP positive.
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1 2 3 4 5 6
Result line
Control line
Fig. 5. Comparison of the dose–response phenomenon of AsAGP by immunochromatography. AsAGP was spiked in the normal sample. Lane 1,
mixed glycoprotein (a1-acid glycoprotein, haptoglobin, a2-macroglobin); Lane 2, normal serum; Lane 3, 1.5 Ag/mL of AsAGP; Lane 4, 2.0 Ag/mL
of AsAGP; Lane 5, 3.0 Ag/mL of AsAGP; Lane 6, 4.0 Ag/mL of AsAGP.
E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123 121
The risk rates about various hepatic diseases were
similar to those of previous results by ELISA (Song
et al., 2003).
4. Discussion
Radioimmunoassay (RIA) and enzyme-linked im-
munosorbent assay (ELISA) are the traditional methods
used for the measurement of some analytes. Although
RIA and ELISA both have the advantages of high
sensitivity, both of the processes are time-consuming
and require a well-trained technician and special equip-
ment in the laboratory. The immunochromatographic
strip method has a number of advantages. To meet
clinical requirements for speed, many types of bedside
assays which used immunochromatographic strips have
been developed. For example, an immunochromato-
graphic strip test for IL-6 was developed by Trebeden
et al. (Trebeden et al., 2001; Kayem et al., 2005). The
detection threshold of IL-6 was 100 pg/mL in vaginal
secretions. In most cases, analytes can be detected at
ranges of nanograms with an immunochromatographic
strip. The immunochromatographic strip test is useful in
an acute disease state, as well as a chronic disease state.
Serum asialo a1-acid glycoprotein (AsAGP) has
been reported as a major component of serum asialo-
Table 1
AsAGP determination by mAb–lectin sandwich ELISA and immunochroma
Clinical diagnosis Total number AsAGP by ELISA
Concentration (Ag/mL)
Normal 29 0.43–1.35
LC (group 1) 25 1.5–2.5
LC (group 2) 32 2.5–3.5
LC (group 3) 16 N3.5
LC: liver cirrhosis.
glycoproteins in patients with hepatic disease (Ser-
bource-Goguel et al., 1983). In our previous clinical
study, serum AsAGP levels were significantly elevated
in 230 patients (3.12+3.42 Ag/mL) with liver cirrhosis
(LC) and 72 patients (3.64+4.01 Ag/mL) with hepa-
tocellular carcinoma (HCC) compared to 97 normal
subjects (0.80+0.29 Ag/mL) (Song et al., 2003). 72%
of patients with liver cirrhosis (LC) and 83% of
patients with hepatocellular carcinoma (HCC) had
serum AsAGP levels higher than the cut-off value
(1.5 Ag/mL). Thus, it was suggested that AsAGP
might be a serum marker for hepatic disease, more
specifically, for LC and/or HCC.
In this study, we have developed an immunochro-
matographic strip for the detection of serum asialo a1-
acid glycoprotein (AsAGP). Through visual recognition
of a result line, this strip test allowed for easy, clear
detection of the AsAGP in serum within 5 min, and the
appearance of the control line verified the validity and
performance of the test. The detection limit of the
immunochromatographic strip that we developed was
1.5 Ag/mL. Actually, a range of AsAGP concentrations
in ng/mL can be detected with high sensitivity. How-
ever, in order to prevent false positive results in normal
serum and screened LC and/or HCC, we optimized the
detection limit at 1.5 Ag/mL.
tographic strips
AsAGP by immunochromatographic strip
Negative (#) % Positive (#) %
27 93 2 7
4 16 21 84
1 3 31 97
0 0 16 100
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Table 2
Serum asialo a1-acid glycoprotein (AsAGP) determination by immu-
nochromatographic strip in patients with hepatic disease
Clinical diagnosis Number Immunochromatographic strip
Negative (#) % Positive (#) %
Normal 31 30 97 1 3
Acute hepatitis 28 16 57 12 47
Chronic hepatitis 40 15 37 25 63
Liver cirrhosis 42 5 12 37 88
Hepatocellular carcinoma 42 4 10 38 90
E.Y. Lee et al. / Journal of Immunological Methods 308 (2006) 116–123122
Even though the immunochromatographic strip test
is useful in an acute disease state, as well as a chronic
disease state, we focused on the detection of liver
cirrhosis and hepatocellular carcinoma rather than
acute hepatitis. In this study, 90% of patients with LC
or HCC showed a positive signal at the result line.
However, approximately 40–50% of patients with hep-
atitis (acute or chronic) were not detected to be AsAGP
positive.
The AsAGP value proportionally increased
according to the progression of hepatic disease in a
serum sample (Song et al, 2003). When the intensity
of the visible color on the result line was measured
by the CS-9300PC scanning densitometer (Shimadzu,
Kyoto, Japan), it proportionally correlated with the
amount of AsAGP present in a sample (data not
shown). Thus, it might be possible that this immu-
nochromatographic strip test can be used for semi-
quantitative evaluation of the serum AsAGP concen-
tration. If this immunochromatographic strip were
connected to a sensor, the risk of acute hepatitis or
chronic hepatitis and the progression of hepatic dis-
ease could be detected.
Taking into account the performance capacity of this
immunochromatographic strip, we then assessed the
practical utility of this one-step immunochromato-
gaphic test. High reproducibility, as well as high sensi-
tivity and specificity, were confirmed with the serum
samples. Even in the presence of various serum glyco-
proteins, such as AGP, haptoglobin, and a2-macroglo-
buline, no interference in the detection of AsAGP was
observed. These results suggest that the immunochro-
matographic test is highly specific for AsAGP. We then
assessed the sensitivity of detection for this immuno-
chromatographic test to compare the concordance of
positive or negative results between the immunochro-
matographic test and the sandwich ELISA method.
Although the concordance between these two methods
was quite high, some discordance between these two
methods was observed in some samples containing
AsAGP at a concentration of 1.50 Ag/mL. This discrep-
ancy may be explained in part by the limitations of the
naked human eye.
In summary, the newly developed immunochroma-
tographic test is simple and reliable for the detection
of AsAGP at serum levels above 1.5 Ag/mL and may
be applicable for point-of-care diagnosis of hepatic
disease.
Acknowledgements
This study was supported by the Korea Ministry of
Science and Technology (M10536080003-05N3608-
00310) and the Small and Medium Business Admin-
istration (SCC1400534). Thanks are due to the staff
of the Catholic University, St. Mary’s Hospital (Dae-
jeon, Republic of Korea) for supplying the serum
samples.
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