mce enzyme immunoassay for carcinoembryonic antigen and alpha-fetoprotein using electrochemical...

Research Article

MCE enzyme immunoassay forcarcinoembryonic antigen and alpha-fetoprotein using electrochemical detection

An MCE electrochemical enzyme immunoassay protocol for the determination of

carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was reported. Two anti-

gens (Ag), CEA and AFP, were incubated simultaneously with an excess amount of

horseradish peroxidase-labeled antibody (Ab*). The free Ab* and the Ab*–Ag complex

produced in the solution were first separated through a postcolumn reaction and then

traced by the enzyme substrate o-aminophenol. The 3-aminophenoxazine produced in

enzyme reaction was detected with downstream amperometric detection. The separa-

tions were performed at a separation voltage of 11.4 kV and were completed in less than

60 s. The better analytical performance and distinct miniaturization/portability for MCE

at less assay time and sample volume consumption was achieved. The detection limit of

CEA and AFP was calculated to be 0.25 and 0.13 ng/mL, respectively. Therefore, MCE

could be used as a sensitive and new tool in separation science and offered considerable

promise in biological sample analysis or quick clinical diagnosis.

Keywords:

Alpha-fetoprotein / Carcinoembryonic / Electrochemical detection / Enzymeimmunoassay / MCE DOI 10.1002/elps.200800805

1 Introduction

Recently, MCE technology has attracted significant attention

and was recognized as a powerful tool for proteomics [1, 2],

clinical and forensic analysis [3], environmental monitoring,

and biological assays [4, 5]. The separation principle of MCE

was the same as conventional CE but offered a number of

advantages, including short detection time, high through-

put, low consumption of sample and reagents, automatic

control, and easy integration [6–10].

Among different detection methods coupled with MCE,

fluorescence detection has been widely used due to its ultra-

high selectivity and sensitivity and the compatibility of

detection mode according to the dimensions of microchips

[11–14]. However, one major drawback of common fluor-

escence detection lies in the need to derivatize most analytes

before analysis. In recent years, the possibility to couple the

CE separation with MS detection through microfluidic chip

has been extensively studied by many researchers. However,

the coupling was difficult due to interface issue, until now

no report deals with practical integration of electrokinetical

separation of proteins and MS detection.

Another detection system that can be coupled to MCE

was electrochemical detection (ED). ED was ideally suited to

miniaturized analytical systems and was an attractive alter-

native detection mode for MCE devices [15–17]. The sensi-

tivity and selectivity of ED were comparable to those of LIF

detection, and many compounds can be directly detected

without derivatization. The combination of MCE and ED

would reduce the detection time and cut down sample and

reagent volume. In addition, the microchip devices possess

inherent advantages, for example, the mixing of the analyte

components in microscale channels through diffusion was

very rapid; also, several assay processes, such as sample

volume metering, mixing, preconcentration and separation,

could be integrated on a chip [18–23]. In 2001, Wang and

co-workers reported a microchip electrochemical enzyme

immunoassays (EIAs) platform. In the microfluidic chip,

immunochemical and enzymatic assays were integrated in

the same microchannel for the detection of glucose and

insulin [24]. Amperometric detection of the analytes could

be accomplished through the oxidation of electroactive

products after precolumn and postcolumn reaction steps.

Electrochemical EIAs, combining antigen–antibody reac-

tions with amperometric detection of the product of the

enzymatic reaction, have evolved dramatically over the past

Shusheng ZhangWei CaoJing LiMingming Su

Key Laboratory of Eco-chemicalEngineering, Ministry ofEducation, College of Chemistryand Molecular Engineering,Qingdao University of Scienceand Technology, Qingdao,P. R. China

Received December 6, 2008Revised February 6, 2009Accepted February 6, 2009

Abbreviations: AFP, alpha-fetoprotein; AP, 3-amino-phenoxazine; BR buffer, Britton-Robinson buffer; CEA,

carcinoembryonic antigen; ED, electrochemical detection;

EIA, enzyme immunoassay; HRP, horseradish peroxidase;

OAP, o-aminophenol; TMB, 3,30,5,50-tetramethylbenzidine

Correspondence: Professor Shusheng Zhang, College of Chem-istry and Molecular Engineering, Qingdao University of Scienceand Technology, Qingdao 266042, P. R. ChinaE-mail: [email protected]: 186-532-84022750

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 3427–3435 3427

two decades [25–28]. MCE enzyme immunoassay electro-

chemical detection (MCE-EIA-ED) could be well suited to

improve immunoassay performances.

As a tumor marker, increased levels of carcinoem-

bryonic antigen (CEA) and alpha-fetoprotein (AFP) in

human serum are associated with certain tumors. So, the

determination of tumor and cancer markers plays an

important role in screening for a disease, in diagnosing of

relating diseases, and in the prognosis of a disease. The

techniques used for quantitative determination of tumor

markers were usually immunoassay, including radio-

immunoassay [29, 30], chemiluminescence immunoassay

[31], enzyme-linked immunosorbent assay [32], fluor-

oimmunoassay [33], and electrochemical immunoassay

[34, 35], of which many studies have been reported.

However, these conventional methods required relatively

large amount of expensive reagents, such as labeled anti-

bodies and relatively long assay times due to several incu-

bation and washing steps needed in detection procedure.

In this paper, we demonstrate advantages of using

amperometry as a detection mode for on-chip EIAs. The

CEA and AFP were incubated simultaneously with two

relating enzyme-labeled antibodies in the sample tube.

Then, the immunocomplexes were injected into the sample

reservoir and separated, and the liberated 3-aminophenox-

azine (AP) product was detected amperometrically at the

end channel by three-electrode system. The MCE system

allowed simultaneous testing for CEA and AFP to be

performed more rapidly, easily, and economically, and

hence it provides an important foundation for the future

development of multianalyte detection on MCE.

2 Materials and methods

2.1 Chemicals and reagents

The CEA and AFP kits were purchased from Biocell

Laboratory (Zhengzhou, China). The CEA-EIA kit consisted

of standards and a solution containing one monoclonal anti-

CEA labeled with horseradish peroxidase (HRP). The AFP-

EIA kit consisted of standards and a solution containing one

monoclonal anti-AFP labeled with HRP. The kits were stored

at 41C. The human serum samples from normal human and

patient with colonic cancer and liver cancer were provided by

the Medical School Hospital of Qingdao University (Qingdao,

China) and stored at -201C. The running buffer consisted of

1.0 mM H2O2 and 1.0� 10�2 M Britton-Robinson buffer

(BR buffer, the mixture of 0.98 g H3PO410.60 g HAc1

0.62 g H3BO3 was diluted to 1 L and adjusted to pH 5.0 with

0.2 M NaOH). A 1.0� 10�2 M stock solution of o-aminophe-

nol (OAP) was prepared by dissolving an appropriate amount

of OAP in water. Unless stated otherwise, all other reagents

were of analytical grade and purchased from local standard

reagent suppliers. All solutions were prepared with double-

distilled water and filtered through 0.45 mm cellulose acetate

membrane filters (Shanghai Yadong Resin, Shanghai, China)

before use.

2.2 Apparatus and conditions

The MCE-EIA-ED system is illustrated in Fig. 1. It consists

of three parts. The ED system used in this work was

purchased from Xi’an REMEX Analyse Instrument (Xi’an,

China), equipped with an electrochemical analyzer (Model

MPI-A) to perform the amperometric detection at a constant

voltage. A programmable high-voltage power supply

containing multiple terminals (Model MPI-A, Xi’an REMEX

Analyse Instrument) provided a variable voltage range of

0–20 kV. A PDMS/glass hybrid chip was provided by Dalina

Institute Chemical Physics, Chinese Academy of Sciences.

ED was carried out with a three-electrode system

consisting of a Pt working electrode, an Ag/AgCl reference

electrode, and a Pt wire auxiliary electrode. The Ag/AgCl

reference electrode and Pt wire auxiliary electrode were

inserted in the detection reservoir. The working electrode

was ‘‘U’’-shaped (length 0.3 mm, width 0.2 mm) manu-

factured through depositing a layer of Pt on the substrate

employing hot vaporizing and vacuum sputtering. The

‘‘U’’-shaped electrode showed an excellent performance for

their maximized working areas and minimized exposed

ones. The electrode pattern was designed according to the

previous literature [36–38], in which all were constructed on

Figure 1. Schematic layout of the MCE-ECDdevice. 1, buffer reservoir (BR); 2, samplereservoir (S); 3, sample waste (SW); 4,detect reservoir (D); 5, substrate reservoir(SR); 6, Pt working electrode; 7, Ag/AgClreference electrode; 8, Pt auxiliary elec-trode. The length of the separation channeland reaction channel was of 35 and 10 mm,respectively.

Electrophoresis 2009, 30, 3427–34353428 S. Zhang et al.


glass microchip, but here we found it much easier

to perform on PDMS/glass hybrid chip. The distance

between channel and working electrode was about

0.1 mm. The magnification images of working electrode and

detection reservoir on PDMS/glass hybrid chip are shown in

Fig. 2.

2.3 MCE-EIA procedures

Microchip channels were rinsed before electrophoresis with

0.1 M NaOH (2 min), double-distilled water (2 min), and

running buffer (10 min) for each analysis. Between runs the

channel was rinsed with buffer for 10 min. The immunoas-

say protocol was a noncompetitive format. The CEA and

AFP standards or serum samples, and the HRP-labeled anti-

CEA antibody and anti-AFP antibody were added to a

sample tube. The solution was incubated for 15 min at 371C

and then diluted with the running buffer. The MCE analysis

was performed at room temperature.

2.4 Electrophoresis procedures

A programmable high-voltage power supply with multiple

terminals was used for electrokinetic injection and electro-

phoretic separation. The schematic diagram of the injection

and separation of chip-CE is shown in Fig. 3. The sample

waste reservoir was left unused, but buffer was added for

balance. Injection and separation were performed through

precise control of the voltage applied to sample, running

buffer, and substrate reservoirs. In the injection period,

10.5 V voltage was applied between sample reservoir and

detection reservoir for 8 s. And then, running buffer,

detection, and substrate reservoirs were floated, 11.4 kV

voltage between buffer reservoir and detection reservoir was

applied to perform the separation. At the same time,

substrate reservoirs were applied with 11.0 kV voltages to

perform substrate injection and maintained during the

whole process of reaction and detection. When the separated

Ab� and Ag–Ab� ran from the separation channel into the

reaction channel, HRP catalyzed the reaction of enzyme

substrate OAP and H2O2. The reaction product, AP, was

amperometrically detected on the Pt working electrode at

the outlet of channel.

Figure 2. The magnification images of working electrode anddetection reservoir on PDMS/glass hybrid chip. The structure ofworking electrode was ‘‘U’’-shaped (length 0.3 mm, width0.2 mm). The distance was about 0.1 mm between channel andworking electrode.

Figure 3. Schematic diagram of the simpleinjection and separation of chip-CE. (A)Cross-channel injector and injection; (B)separate operation; (C) pattern of MCE.Ab-E, enzyme-labeled antibody; Ag, anti-gen; 1, buffer reservoir (BR); 2, samplereservoir (S); 3, sample waste (SW); 4,detect reservoir (D); 5, substrate reservoir(SR).

Electrophoresis 2009, 30, 3427–3435 Microfluidics and Miniaturization 3429


2.5 Preparation and analysis of human serum samples

Preparation and analysis of human serum samples

was performed according to the literature [39]. Into six

single-analyte samples consisting of different concentra-

tions of antigens were added fixed amount of monoclonal

HRP-labeled antibodies. All mixtures were incubated at

371C for 15 min. Each sample was analyzed in triplicate.

The calibration curve was established by plotting the peak

area of the complex versus the final concentration of

antigens.

For simultaneous measurements, CEA and AFP antigen

standards were added into normal human serum to simu-

late patient serum containing both antigens. Immunocom-

plex was formed in serum samples containing CEA and

AFP in the sample tray and sample incubation temperature

was programmed for 371C. The MCE analysis was

performed at room temperature. Each sample was analyzed

in triplicate.

3 Results and discussion

3.1 Electrochemical behavior of AP

HRP can catalyze the oxidation of OAP to AP by H2O2,

as shown in Scheme 1. The electrochemical behavior

of AP on a glassy carbon electrode in 5.0� 10�2 M

Tris-HCl buffer (pH 6.0) has been studied in our

previous report [40]. Here, the electrochemical behavior of

AP was reinvestigated at different ED system on a Pt

electrode in 1.0� 10�2 M BR buffer (pH 5.0) using cyclic

voltammetry. The cyclic voltammogram is shown in

Fig. 4, which is similar from the detect reservoir to the

beaker. It was found that AP could be reduced on Pt

working electrode below �0.46 V (versus Ag/AgCl) [41]. At

the potential, the detected current depended only on the

reduction of AP, which was not proportional to the

concentration of AP. Therefore, the activity of HRP

could be measured by detecting the reduction current of

AP. These demonstrated that the method of electrochem-

istry detected using Pt microelectrode on glass substrate is

feasible.

3.2 Factors influencing MCE-EIA

The pH of the buffer solution has an important effect on the

surface characteristics, EOF of the uncoated PDMS chip,

and the effective electric charge of the ion. The free Ab� and

Ag–Ab� immunocomplex can achieve a baseline separation

in the pH range from 4.5 to 6.0 and separation time

increased as pH decreased and the best separation efficiency

was realized at pH 5.0. Thus, the pH 5.0 was chosen for the

MCE separation. The electropherograms of Ab� and

Ab�–Ag immunocomplex for CEA (A) and AFP (B) with

different pH are shown in Fig. 5.

The running buffer concentration (Cb) is another

important parameter. The effect of the running buffer

concentration on tm, ip, W1/2, and N was studied ranging

from 5.0� 10�3 to 0.1 M, and are listed in Table 1. The

separation time and separation efficiency increase with an

increase in the concentration of buffer solution. However, a

relative low ip was observed with increasing Cb due to larger

Joule heating. A relative low concentration of running buffer

was used to decrease the Joule heating, which also caused

OH

NH 2

+ 3 H 2O2

HRP

O

N NH 2

O

OAP AP

+ 6 H2O2

O

N NH 2

O O

HN NH 2

OH

+ + 2e2H +

Scheme 1.

Figure 4. The cyclic voltammogram curves of 2.8�10�4 M AP ata Pt electrode in 1.0�10�2 M BR buffer (pH 5.0). Scan rate is100 mV/s. 1, in the detect reservoir on chip; 2, in the beaker.



immunocomplex dissociation [42]. The 1.0� 10�2 M BR

buffer was selected for separation.

The separation voltage is also an important parameter.

The influence of the applied separation voltage upon the

analytical performance was examined over the 11.0–1.5 kV

range. The electropherograms of Ab� and Ab�–Ag mixtures

for CEA (A) and AFP (B) with different separation voltage

are shown in Fig. 6. The results indicated that such voltage

is sufficient for effective immunological and enzymatic

reactions and the subsequent separation of the anti-

body–antigen complex from the free antibody. As expected,

increasing the separation voltage over the 11.0–1.5 kV

range decreased the migration time and increased separa-

tion velocity for the free antibody and its complex with

antigen. Higher field densities shorten the contact/reaction

time of the separated immunocomplex with substrates in

the postcolumn, thus impairing the efficiency of the enzy-

matic reaction. The separation voltage of 11.4 kV was used

for all subsequent work, as it provided the most favorable

balance between speed, sensitivity, resolution, and isolation

from the detection circuitry.

In amperometric detection, Ed, the voltage applied to the

working electrode directly, affects the sensitivity, detection

limit, and stability of this method. The effect of Ed on the ipwas studied ranging from �0.65 to �0.35 V. With the Ed

decreased, the ip increased along with a larger noise. When

the Ed was �0.50 V, the working electrode showed a good

stability, high reproducibility, acceptable background

current, and the highest S/N ratio.

The time and voltage of sample injection had a great

influence on the sample injection volume and thus ip and

peak area. Too small volume injected would lead to low or

even no signal, while a heavy peak trail could be induced by

too large injection volume. Hence, sample injections were

usually performed by applying a voltage of 10.5 kV for 8 s to

the sample reservoir. The curve on influence of the time and

voltage of sample injection are shown in Fig. 7.

3.3 Calibration curves for CEA and AFP

In this method, the noncompetitive format was performed

on a microchip electrochemical EIAs platform. Ag reacted

Figure 5. Electropherograms of Ab� andAb�–Ag mixtures for CEA (A) and AFP (B)with different pH of the buffer. (1) 4.5; (2)5.0; (3) 5.5; (4) 6.0. CH2O2

: 1.0� 10�3 M; COAP:1.0�10�3 M; CBR: 1.0�10�2 M; Ed: �0.5 V;separation potential: 11.4 kV; injectionpotential and time: 10.5 kV and 8 s.

Table 1. The values of tm, ip, W1/2, and N at different running

buffer concentrationa)

Cb (10�3 M) tm (s) ip (nA) W1/2 (s) 103N

5 8 10.2 5.6 12.4

10 20 17.5 5.9 13.8

20 50 18.2 6.0 19.1

40 100 16.0 6.2 22.8

80 120 16.0 6.4 24.7

100 200 14.7 7.2 25.1

a) Running buffer, BR (pH 5.0); COAP, 1.0�10�3 M; Ed, �0.5 V;

separation voltage, 11.4 kV; injection voltage and time, 1

0.5 kV and 8 s.



with an excess amount of the solution containing one HRP-

labeled monoclonal Ab� from the EIA kits. The immune

reaction proceeded in liquid phase, avoiding repetitive

incubation and washing steps in solid phase. The conditions

of the immune reaction were controlled according to the

procedure recommended by conventional ELISA, and

the commercial CEA and AFP kits were used directly. The

3,30,5,50-tetramethylbenzidine (TMB) spectrophotometric

ELISA assay was performed following the manufacturer’s

procedure.

For the samples, each of which independently containing

CEA and AFP, the relationship between the immunocomplex

peak areas and the antigen concentrations was investigated

under optimal conditions. The typical electropherograms

obtained in this research for the separation of free antibody

and its immunocomplex with increasing concentrations of

CEA and AFP antigens are shown in Fig. 8, respectively. Two

peaks appeared in the electropherograms, corresponding to

Ab� (peak 1) and Ag–Ab� (peak 2). With the increase of the

antigen concentrations, peak 1 decreased and peak 2

increased correspondingly. The antibodies are bidentate,

capable of binding to two antigen molecules. When the

antibody was in excess of antigen, the formation of binary

complex (1:1 stoichiometry) was favored. Only when the

binding sites are limited, as in the case of lower antibody

concentration, does the tertiary complex (1:2 stoichiometry)

dominated [43]. The antibody concentrations used in our

experimental was much higher than those of the antigens,

thus the binary complex dominated.

The calibration curves for the two antigens can be

acquired by plotting the peak area against the concentra-

tions of CEA and AFP antigens, respectively. The linear

range (n 5 7, r 5 0.9962) and detection limit for CEA were

Figure 6. Electropherograms of Ab� andAb�–Ag mixtures for CEA (A) and AFP (B)with different separation voltage. (1) 1

1.5 kV; (2) 11.4 kV; (3) 11.2 kV; (4) 11.0 kV.Other conditions are same as in Fig. 5.

200

0

5

10

15

20

25

i p (

nA)

injection voltage (V)

42

4

6

8

10

12

14

16

18

20

22

i p (

nA)

t (s)

6 8 10 12

300 400 500 600 700 800

Figure 7. The curve of influence for the time and voltage ofsample injection. CH2O2 : 1.0� 10�3 M; COAP: 1.0� 10�3 M; CBR:1.0�10�2 M (pH 5.0); Ed: �0.5 V; separation potential: 11.4 kV.



0.5–66.0 and 0.25 ng/mL, with the equation of linear

regression being q 5 1.411410.3236C (q is the peak area

(nC) and C is the concentration of CEA antigen (ng/mL)).

CEA (8.0 ng/mL) was measured by 11 parallel points, with

determined RSD of 4.2%. The detection limit with TMB

spectrophotometric ELISA method was 5.0 ng/mL, which

was 20 times lower compared with that of the TMB spec-

trophotometric ELISA method.

The linear range (n 5 9, r 5 0.9977) for AFP was from

0.5 to 80.0 with a detection limit of 0.13 ng/mL and the

equation of linear regression being q 5 0.848610.3583C(q is the peak area (nC) and C the concentration of AFP

antigen (ng/mL)). AFP (10.0 ng/mL) was measured by 11

parallel points, with determined RSD of 3.8%. The detection

limit of this method was eight times lower compared with

that of the TMB spectrophotometric ELISA method. This

method offered several advantages such as short analysis

time (20 s), low reagent consumption, and simple operation

compared with those of spectrophotometric ELISA method.

3.4 Detection of CEA and AFP in human sera

Blood samples from normal human, alimentary tract

patient, and patient with liver cancer were analyzed by

this assay. Blood samples were first diluted and a fixed

amount of monoclonal HRP-labeled antibodies was added.

Then, all mixtures were incubated at 371C for 15 min. The

representative electropherograms for normal human and

infected human serum are shown in Fig. 9. There were

Figure 8. Electropherograms of Ag andAb�mixtures for CEA (A) and AFP (B) withdifferent concentrations. A, concentrationof CEA (ng/mL): (1) 0, (2) 5.5, (3) 22.0,(4) 44.0, (5) 66.0; B, concentration of AFP(ng/mL): (1) 0, (2) 10.0, (3) 18.0, (4) 28.0,(5) 50.0. CH2O2

: 1.0�10�3 M; COAP:1.0�10�3 M; CBR: 1.0�10�2 M (pH 5.0);Ed: �0.5 V; separation potential: 11.4 kV;injection potential and time: 10.5 kV and8 s.

Figure 9. Typical electropherograms fornormal and infected human serum samplesCEA (A) and AFP (B). (1) Normal humanserum samples and (2) infected humanserum samples. Other conditions are sameas in Fig. 8.



small peaks for the immunocomplex for normal human.

For positive samples, obvious peaks at the position

corresponding to the immunocomplex peaks were observed.

To compare with the results with routine TMB ELISA tests,

the materials and immunoreaction steps in our assay were

partly the same as that in the ELISA methods. The results

were corresponding to each other, indicating the high

feasibility of the presented method.

3.5 Simultaneous detection of CEA and AFP in

human serum

Simulated samples containing CEA and AFP antigen

standards were analyzed by this method and illustrated in

Fig. 10. It was shown that the two analytes could be detected

within 60 s, indicating a fast detection. Three repetitive

experiments were made under the same conditions. The

analytical results were presented in Table 2. The results of

CEA and AFP obtained by MCE-EIA and spectrophoto-

metric ELISA methods for comparison are listed in Table 3.

The calibration curves for the two antigens could be

acquired by ELISA method against MCE method, respec-

tively. The equation of linear regression for CEA is

y 5 1.896010.85185x (y is the MCE methods, x the ELISA

methods, r 5 0.9956 n 5 6). The equation of linear regres-

sion for AFP is y 5�0.227110.9748x (y is the MCE

methods, x the ELISA methods, r 5 0.9986 n 5 6).

4 Concluding remarks

A protocol using microfabricated device that integrates

multiple steps of electrochemical EIAs on a chip platform

was successfully established, which was applied to detect

CEA and AFP in human serum samples. Such an on-chip

integration of enzymatic reactions, electrophoretic separa-

tion, and amperometric detection allowed performing

immunoassays more rapidly, easily, and economically. The

main challenge in the development of this assay was to

establish separation conditions. The concept of microchip

electrochemical EIAs has been illustrated using the CEA

and AFP as models. It could be readily extended to a broad

variety of analytes of clinical, environmental, or biotechno-

logical significance. The new MCE electrochemical immu-

noassay strategy offers a considerable promise for designing

self-contained and disposable chips for decentralized clinical

or on-site environmental testing.

This work was supported by the National Natural ScienceFoundation of China (No. 20775038) and the Natural ScienceFoundation of Shandong Province (No. Z2007B31).

The authors have declared no conflict of interest.

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Figure 10. Electropherogram of simulated human serumcontaining CEA (22.0 ng/mL) and AFP (28.0 ng/mL). (1) Anti-AFP-HRP; (2) anti-CEA-HRP; (3) AFP-anti-AFP-HRP; (4) CEA-anti-CEA-HRP. Other conditions are same as in Fig. 8.

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Antigens Sample

content

(ng/mL)

Added

(ng/mL)

Detection

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RSD

(%, n 5 3)

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AFP 10.8 20.0 31.8 3.7 101.5

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This method ELISA method This method ELISA method

(ng/mL) (ng/mL) (ng/mL) (ng/mL)

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3 11.43 12.10 12.24 11.84

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5 24.36 23.19 26.26 25.32

6 29.30 27.12 32.85 31.97

a)Represents no signal.



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