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: eysong@kribb.re.kr (E.Y. Song).

ess: Food and Drug Division, Ulsan Institute of Health and Environment, Yaum, Ulasan 576-10, Korea.

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

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

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-

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.

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

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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