ORIGINAL PAPER

Development of a sensitive micro-magneticchemiluminescence enzyme immunoassayfor the determination of carcinoembryonic antigen

Wijitar Dungchai & Weena Siangproh & Jin-Ming Lin &

Orawon Chailapakul & Si Lin & Xitang Ying

Received: 8 August 2006 /Revised: 28 September 2006 /Accepted: 2 October 2006 / Published online: 18 November 2006# Springer-Verlag 2006

Abstract A micro-magnetic chemiluminescence (CL) en-zyme immunoassay with high sensitivity, selectivity, andreproducibility was developed for the determination of thetumor marker, carcinoembryonic antigen (CEA) in humanserum. A sandwich scheme assay has been utilized withfluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CLsignal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was directly proportional to theamount of analyte present in a sample solution. Theinfluences of the reaction time of antigen with antibody,the reaction time of substrate with label, the dilution ratio ofALP-labeled anti-CEA antibody, the concentration ofFITC-labeled anti-CEA antibody, and other relevant vari-ables upon the CL signal were examined and optimized.

The CL responses depended linearly on the CEA concen-tration over the range from 2 to 162 ng mL−1 in alogarithmic plot. Assay sensitivity as low as 0.69 ngmL−1 was achieved. A coefficient of variance of less than13% was obtained for intra- and inter-assay precision. Thismethod has been successfully applied to the analysis ofCEA in human serum. According to the procedure based onspiked standards, the recoveries obtained were 80–110%.Comparison experiments were carried out with the com-mercially available CEA chemiluminescence immunoassay.Satisfactory results were obtained according to a pairedt-test method (t value< tcritical at the 95% confidence level).

Keywords Micro-magnetic beads .

Carcinoembryonic antigen . Tumor .

Chemiluminescence enzyme immunoassay .Marker

Introduction

The determination of tumor markers plays an important rolein clinical research and diagnosis. Currently, tumor markersare widely used to provide both an indication of response totherapy and disease progression or recurrence in patientswith cancers [1, 2]. Carcinoembryonic antigen (CEA) is ahighly glycosylated cell surface glycoprotein with amolecular weight of 180–200 kD, which occurs in highlevels in colon epithelial cells during embryonic develop-ment. Levels of CEA are significantly lower in colon tissueof adults, but can become elevated when inflammation ortumors arise in any endodermal tissue, including in thegastrointestinal tract, respiratory tract, pancreas, and breast[3–5]. An overexpression of CEA protein has been detectedin a variety of adenocarcinomas, including gastric, pancre-atic, small intestine, colon, rectal, ovarian, breast, cervical,

Anal Bioanal Chem (2007) 387:1965–1971DOI 10.1007/s00216-006-0899-y

W. Dungchai :W. Siangproh : J.-M. Lin (*)The Key Laboratory of Bioorganic Phosphorus Chemistry &Chemical Biology, Ministry of Education,Department of Chemistry, Tsinghua University,Beijing 100084, Chinae-mail: jmlin@mail.tsinghua.edu.cn

W. Dungchai :W. Siangproh :O. ChailapakulDepartment of Chemistry, Faculty of Science,Chulalongkorn University,Patumwan,Bangkok 10330, Thailande-mail: corawon@chula.ac.th

S. Lin :X. YingBeijing Chemclin Biotech Co., Ltd.,Beijing Academy of Science and Technology,Haidian,Beijing 100094, China

(*)

and non-small-cell lung cancers. Carcinoembryonic antigenis also expressed by epithelial cells in several non-malignant disorders, including diverticulitis, pancreatitis,inflammatory bowel disease, cirrhosis, hepatitis, bronchitis,and renal failure and also in heavy smokers [6–9].Therefore CEA should not be regarded as a tumor-specificmarker for the screening of undetected cancers in thegeneral population; nevertheless, the determination ofcarcinoembryonic antigen levels provides important infor-mation about patient prognosis, recurrence of tumors aftersurgical removal, and effectiveness of therapy.

Immunological assays are one of the most importantmethods in the field of clinical diagnoses and biochemicalstudies because of their extremely high selectivity andsensitivity. Immunological methods are the analyticaltechnique of choice for the quantitative determination ofCEA. The radioimmunoassay was the first developedmethod for circulating CEA in 1969 [10]. Even thoughradioimmunoassay was the most commonly used methodfor antigen detection [11], radioactive labels are potentiallyharmful to the operators. Hence, enzyme-linked immuno-sorbent assays (ELISAs) [12], piezoelectric immunoassay[13], chemiluminescence (CL) assay [14–16], colorimetricimmunoassay [17], fluoroimmunoassay [18, 19], andliposome immunoassay [20] have been adapted to clinicalanalysis. Unfortunately, most of these methods involveseparation steps and/or require highly qualified personal,extended assay times, or sophisticated instrumentation.Electrochemical immunosensors [21–24] have become thepredominant analytical technique for CEA detection,although these methods are thus far not available ascommercial assay kits.

As small increases or decreases in tumor marker levelscan be indicative of disease progression or early recurrenceof disease, fluctuations in tumor marker levels can result infalse positive or false negative assay values in a givenpatient. Although the above mentioned traditional methodshave low detection limits and are suitable for the determi-nation of CEA in serum, a sensitive and rapid method forthe analysis is still needed.

Our ultimate goal in this research is to provide ascreening device for fast analysis with low limits ofdetection and high accuracy to prevent false negatives andpositives so as to maintain confidence in the monitoringsystem. To this end, a small volume microbead-basedimmunoassay with CL detection is being developed forthe detection of CEA in human serum. The mainadvantages of bead-based immunoassay include increasingthe surface area for immobilization of antigen or antibodyand reducing the incubation time [25, 26]. Focus wasplaced on CL because we aim to develop a bead-basedassay for use in commercial CEA test kits. Herein, wereport the preliminary results on using bead-based chemi-

luminescence immunoassay (CLIA) for the determinationof CEA in standard chemical form and human serum. Theperformance characteristics of bead-based CLIA arereported.

Experimental

Instrumentation and chemicals

A universal luminometer (Chain-based flash glow devicefrom Berthold Technologies GmbH & Co. KG, Germany),sample tubes (with dimensions of 12-mm diameter×60-mmlength), and a CL micro-plate reader (BHP9504 fromHamamatsu Co., China) were used. The incubation proce-dures were carried out at a constant temperature of 37 °C(HHW 21.CR 600, China). The shaker (ZXWL-100, China)was used to shake the solution after adding the micro-magnetic beads. After the enzymatic reaction, the tubes canbe directly placed into the luminometer for the measure-ment. Data acquisition and treatment were performed withan integrated 16-bit microprocessor system.

The immunomagnetic microbeads (3-micron, 0.1% w/v)coated with anti-fluorescein isothiocyanate antibody (FITC)were purchased from Adaltis (Shanghai, China). Thesolution of FITC-labeled monoclonal anti-CEA antibody(500 μg mL−1), the standard solutions of carcinoembryonic(CEA), and alkaline phosphate-labeled carcinoembryonicantibody (ALP-labeled anti-CEA antibody) were obtainedfrom Beijing Chemclin Biotech Co., Ltd. (Beijing, China).4-Methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD) was purchased from Diagnos-tic Products Corporation (California, USA). Bovine serumalbumin (BSA) and sodium azide were from Merck(Darmstadt).

Buffers

The coating buffer for microbeads consisted of 0.1 mol L−1

Tris-HCl buffer, 0.1% (w/v) BSA, and 0.1% sodium azide.The washing solution was 0.1 mol L−1 Tris-HCl buffer(pH 7.4) and 0.1 mol L−1 NaCl. The solution of 0.1 mol L−1

Tris-HCl buffer (pH 7.4) containing an appropriate amountof BSA was used as dilution buffer for FITC-labeled anti-CEA antibody and ALP-labeled anti-CEA antibody. The CLsubstrate buffer was 0.1 mol L−1 Tris-HCl (pH 9.5) with1 mmol L−1 MgCl2 and 0.02% (w/v) NaN3.

Immunoassay procedure

First, 25 μL of carcinoembryonic antibody or serumsamples was placed into the test tube. Then, 50 μL dilutedALP-labeled anti-CEA antibody and 50 μL diluted FITC-

1966 Anal Bioanal Chem (2007) 387:1965–1971

labeled anti-CEA antibody were added stepwise andincubated for 1 h at 37 °C. After the sandwich reaction,50 μL coated anti-FITC-labeled magnetic beads were addedand incubated with shaking for another 5 min. Then, themagnetic plate separation was inserted under the test tubesfor bead separation (2 min). Antibody-coated beads and anyspecific captured material were attracted by the magnets tothe bottom of the test tubes and unwanted materials areremoved from the test tubes. The test tubes were thengently tapped against tissue paper to remove all fluid.During the washing steps, the washing solution was addedinto the test tubes by placing them outside the magneticplate, so the particles were suspended in the washingsolution. Washing was performed with 1.0 mL of thewashing solution three times. Finally, 300 μL CL substratesolution was added, the mixture was incubated for 20 minat room temperature (in the dark), and the emitted photonswere measured.

Micro-plate chemiluminescence enzyme immunoassay(micro-plate CLEIA)

The immunoassay procedure was reported in detail on theproduct label. Briefly, 50 μL of antigen was added into themicrowell. After that 50 μL of anti-CEA antibody wasadded and thoroughly mixed for 30 s. Then, the microplateswere allowed to stand sealed at 37 °C for 1 h. Fivewashings were performed, after which 50 μL of CLsubstrate was added, the mixture was incubated (in thedark) at room temperature for 30 min, and the emittedphotons were measured.

Data analysis

Standard and samples were measured, and CL intensityvalues were integrated. Standard curves were obtained byplotting the logarithm of CL intensity (in relative lightunits, RLUs) against the logarithm of analyte concentrationand fitting to a linear equation.

Human serum

Normal human sera obtained from a hospital in Beijing,China, were spiked with CEA in different concentrations.All samples were analyzed using the micro-plate and micro-magnetic CLEIA without any pretreatment.

Statistical analysis

For comparing two measurement systems that are supposedto be equivalent, results were tested by paired t-test andBland-Altman model. The methods have been described indetail elsewhere [27, 28].

Results and discussion

Optimization of immunoassay reagents

The immunoreaction reagent is a key parameter affectingthe sensitivity of immunoassay. In general, the minimumamounts of reagents that maintained acceptable sensitivitywere used to reduce the assay cost. Therefore, in thisexperiment, the dilution ratios of ALP-labeled anti-CEAantibody and the concentrations of FITC-labeled anti-CEAantibody were studied. As shown in Fig. 1, when thedilution ratios of ALP-labeled anti-CEA antibody wereincreased from 1:5,000 to 1:500, the RLU responses alsoincreased at all examined concentrations of FITC-labeledanti-CEA antibody (use the standard CEA concentration of162 ng mL−1). Hence, a 1:500 dilution ratio of ALP-labeledanti-CEA antibody was selected because this ratio providedthe highest RLU intensity. Moreover, significant differenceswere observed between results at comparison results at1:500 and 1:1,000. For the concentration of FITC-labeledanti-CEA antibody, it was found that the signals increaserapidly upon increasing the concentration between 0.1 and0.5 μg mL−1 and levels off thereafter (found in all dilutionratios of ALP-labeled anti-CEA antibody). Hence, aconcentration of 0.5 μg mL−1 FITC-labeled anti-CEAantibody was chosen.

For this immunoassay procedure, the diluted ALP-labeled anti-CEA antibody and diluted FITC-labeled anti-CEA antibody were normally added into the test tubes stepby step. Mixing two antibodies before adding into the testtubes may reduce the analysis time and the assay errorresulting from pipetting. Hence, the calibration curve was

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Fig. 1 Relationships between concentration of FITC-labeled anti-CEA antibody and the dilution ratios of ALP-labeled anti-CEAantibody on the RLU values, using the standard CEA concentrationof 162 ng mL−1. The five curves correspond to the different dilutionratios of ALP-labeled anti-CEA antibody (i.e., 1:500, 1:1,000,1:2,000, 1:3,000, and 1:5,000)

Anal Bioanal Chem (2007) 387:1965–1971 1967

used to study the optimization of the immunoassayprocedure. The results showed that the obtained calibrationcurves were the same with the two procedures (data notshown). Thus, the mixing procedure was employed for thesubsequent experiments.

Physicochemical parameter optimization

The experimental parameters including incubation time andincubation volume were studied with two goals: (1) toimprove immunoassay sensitivity and (2) to study immu-noassay performance under the optimal conditions. Theseexperiments were done using the proposed method de-scribed above.

Effect of immunoassay incubation time

From our previous experience we knew that the time givento the immunoreagents to interact (i.e., the incubation time)may have a direct effect on the sensitivity of theimmunoassay. We therefore varied the length of immuno-reaction time from 30 to 120 min. Figure 2a shows theresults from the assay performance as a function of theincubation time. The intensity increased with longerincubation time. We chose an incubation time of 60 minbecause the signal seemed to stable and no significancedifferences were observed for higher times. In addition, thesensitivity of an incubation time of 60 min is much higherthan at 30 min.

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aFig. 2 Physicochemical param-eter optimization. a Influence ofimmunoassay incubation timeon CL intensity. The antigen andantibody were incubated at dif-ferent time (between 30 and120 min). b Effect of the lengthreaction time between CL sub-strate and ALP-labeled anti-CEA antibody. c Effect of theshaking time to capture effi-ciency between coated micro-magnetic beads and sandwichreaction. d Influence of thevolume of magnetic beads onthe CL response. e Effect ofsubstrate volume on the bead-based chemiluminescence en-zyme immunoassay response

1968 Anal Bioanal Chem (2007) 387:1965–1971

Influence of reaction time between AMPPD CL substrateand ALP-labeled anti-CEA antibody

The results are shown in Fig. 2b. It can be seen that theRLU increased with the incubation time in the range of 5–20 min at all studied volumes (200, 300, and 400 μL). After20 min, the RLU signal did not change. This phenomenoncan be explained by using the fact that the half-life ofluminescence product (AMP-D) is between 2 and 30 mindepending on the environment; after that time, it will bedecomposed, giving the steady state of CL signal. Hence,20 min was selected for the reaction time of substrate andALP-labeled anti-CEA antibody.

Effect of shaking time on capture efficiency

In this system, the immunomagnetic beads coated with anti-FITC antibody are added into the solution containingantigen to provide the binding. The shaking time ofsolution would therefore affect the capture efficiency. Theshaking time was investigated within the range of 5–30 min. The experiment showed (Fig. 2c) that the CLintensity increased with longer shaking times. No signif-icant differences were observed for RLU value between5–15 min. This means that 5 min was suitable enoughtime for the capture, and analysis time was also reduced.Thus, a shaking time of 5 min was set for all subsequentexperiments.

Effect of volume of magnetic beads

The quantity of magnetic beads was critical for the CLintensity. Conditions suitable for monolayer dispersion inthe bottom of the tube were needed. Hence, we studied theeffect of the volume of the solution of magnetic beads onCL intensity. The results showed that the CL intensityincreased with the volume of the solution containingmagnetic beads up to 50 μL, whereas the intensitydecreased above 50 μL, as shown in Fig. 2d. Hence,50 μL of magnetic beads was selected as optimalconditions.

Effect of substrate volume in the bead-based CLEIA

Substrate sensitivity refers to the signal intensity producedby a unit of enzyme activity. Thus, in this experiment, thevolumes of substrate were studied from 100 to 500 μL. Wefound that the CL intensity increased with larger volume ofthe solution over the examined range. We did not obtain theexpected sigmoidal behavior with a defined plateau.Therefore, the ratios (S/S0) of the mean RLU signals with(S) and without (S0) substrate were calculated as a functionof volume. The S/S0 ratio reaches a maximum value at

300 μL (Fig. 2e). Hence, this volume was selected for allsubsequent experiments.

Non-specific adsorption

The polystyrene tubes were used as a container for theimmunoassay reaction. This kind of tube showed highadsorption affinity, so the effect of non-specific adsorptionon the surface of the tube was also investigated. Differentconcentrations of CEAwere used to study the effect of non-specific adsorption on the proposed method. The experi-ment showed (Table 1) that the CL intensity in the absenceof the solution containing magnetic beads did not changewhile the CEA concentrations were increased. On the otherhand, the RLU intensity of the solution containing magneticbeads increased with increasing CEA concentrations.

Linearity-dilution effect

Linearity-dilution effect was studied to check the dilutioneffect of sample with the other solutions to provide anaccurate quantitation. Human serum samples may bediluted with a different solution (e.g., cow serum) in orderto get the concentration within the examined range. Thecalibration curve was used to evaluate this effect. In thisexperiment, we selected cow serum as a diluent solution. Itwas found that the relationship between the concentrationof diluted CEA and the dilution factors gave a high linearitywith correlation coefficient, r=0.9997, as shown in Fig. 3a.This meant there was no effect between the human serumand cow serum in the case of high levels of CEA; we cantherefore use cow serum to dilute samples before analysis.

Calibration and sensitivity

A dose–response curve obtained with the CL detection ofantigen-antibody reaction under optimal conditions isdisplayed in Fig. 3b. Linearity was obtained in the rangefrom 2 to 162 ng mL−1. The detection limit or minimumdetectable limit was defined as the mean RLU signal (S0) atzero concentration plus two times the standard deviation

Table 1 Studies of non-specific adsorption

CEA conc.(ng mL−1)

CL intensity withoutmagnetic beads (RLU)

CL intensity withmagnetic beads added(RLU)

0 7,915 13,2012 7,430 31,6526 7,580 56,69318 6,137 137,34654 10,937 325,130162 10,807 918,547

Anal Bioanal Chem (2007) 387:1965–1971 1969

(SD) (LOD=S0+2SD). The detection limit of CEA wascalculated by obtaining the average RLU signals for 10replicates of zero standard and then adding 2SDs of theaverage. The RLU signals plus two SDs were thenextrapolated from the log–log standard curve and representthe sensitivity of the assay. The value of detection limit forCEA was 0.69 ng mL−1.

Precision

The importance of the precision has often been stressedusing bead-based CL-EIA for quantitative analysis. Theintra-assay precision of the analytical method was calculat-ed by analyzing each concentration ten times per run in1 day. Three different concentration levels were used forthis study. Similarly, these methods were analyzed ten timesat different times, each using two test tubes, to determineinter-assay precision. It can be observed that intra- andinter-assay coefficients of variation were in all cases below

13%. The results of testing are listed in Table 2. Theseresults implied that the proposed method exhibited a highreproducibility.

Quantitative determination of CEA in human serum:comparison with a commercially available CEA CL-EIA kit

The proposed method was applied to determine CEA inhuman serum. We examined the accuracy of the method forthe determination of CEA in human serum by using the

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Table 2 Precision of the micro-magnetic CL-EIA

Assay type Conc. of CEA(ng mL−1)

CV (%)

Intra-assay (n=10) 6 8.718 5.954 4.9

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method of calibration. The linear least-squares calibrationplot had a slope of 0.7408 RLU ng mL−1 and a correlationcoefficient of 0.9994. Before analysis, the normal humansera were spiked with CEA. Recoveries of added CEAranged between 80% and 110%.

In addition, CEA concentrations in normal human serumand the spiked human serum samples were simultaneouslydetermined by using the commercially available CEA CL-EIA kit. The results are shown in Fig. 4. It can be seen thatthe data are tightly scattered about the calibration lines inboth cases. This means that the measurements are compa-rable and acceptable. Moreover, to make sure, the paired t-test was used to evaluate these two immunoassay methods.No significant difference was found at the 95% confidencelevel (t value is less than tcritical). Thus, the analyzed valuesof CEA in human serum can be accepted.

Conclusions

A highly sensitive, specific, and reproducible micro-magnetic CL-EIA has been developed and its feasibilityhas been demonstrated for the determination of CEA inanalyte samples. The proposed assay comprises anti-FITCantibody-coated magnetic beads, FITC-labeled anti-CEAantibody, ALP-labeled anti-CEA antibody, and a CLdetection system. With the assay, CEA can be reproduciblydetected in human serum at a concentration as low as0.69 ng mL−1 even though a short incubation time wasmaintained. This concentration is about 50% lower than thecommercial ELISA test kit (1 ng mL−1 for ELISA) [29, 30].In addition, our assay uses magnetic bead particles and isperformed in a total volume of 300 μL, making it is ideallysuited for high-throughput and robotics-based assay for-mats, for large-volume detection. A stable calibration curvewith a wide dynamic range was also established. Thecalibration curve was linear from 2 to 162 ng mL−1. For ourparticular application, the dynamic range of the assaycovers all CEA levels used for evaluating human cancerousserum. If the RLU reading is higher than or equal to thehighest point of the linear portion, the unknown samplescan be diluted for a proportional measurement with diluentsolution. Data from the developed method highlight theacceptable intra-assay (less than 10%) and inter-assay (lessthan 20%) precision and accuracy (80–110% recovery). Insummary, this assay provides good advantages and couldeasily be accommodated into a variety of detection systemscurrently available on the market.

Acknowledgements The authors gratefully acknowledge the finan-cial support from the National Natural Science Foundation of China

(Nos. 20437020, 20575008) and the Program for Changjiang Scholarsand Innovative Research Team in University (No. IRT0404). WDacknowledges a fellowship from the Thailand Research Fund throughthe Royal Golden Jubilee Ph.D. Program (Grant No. PHD/39/2548).

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