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Carbohydrate antigen 72-4 (CA72-4) was proposed by. Colcher in 1981. It is a tumor-associated glycoprotein. (MW>1000 ku) recognized by monoclonal ...
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Microplate chemiluminescence enzyme immunoassay for the quantitative evaluation of carbohydrate antigen 72-4 in human serum JIN Hui1,2, WANG Xu2†, XIN TianBing1,2, GAO Peng3, LIN Jin-Ming2† & LIANG ShuXuan1 1

College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China; Department of Chemistry, Tsinghua University, Beijing 100084, China; 3 Beijing Chemclin Biotech Co., Ltd., Beijing 100094, China 2

A highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) was developed for the quantitative evaluation of carbohydrate antigen 72-4 (CA72-4) in human serum, using luminol-H2O2 catalyzed by horseradish peroxidase (HRP) as the chemiluminescence system. The simple and quick determination was accomplished through a sandwich reaction mode. Several physicochemical parameters of the immunoreaction, including incubation conditions, antibody coating conditions, dilution ratio of anti-CA72-4-HRP conjugate, and chemiluminescence reaction time, were studied and optimized. The proposed method exhibited a linear range of 0―200 U/mL with correlation coefficient and detection limit of 0.9995 and 0.18 U/mL, respectively. The inter-assay and intra-assay coefficients of variation (CV) were both less than 10%. The average recovery of two clinical sera with low and high concentration CA72-4 was 99.3% and 98.7%, respectively. Normal tumor markers, including AFP, CEA, CA24-2, CA19-9 and CA15-3, did not cross-react with each other. The method’s stability was evaluated by assessing its analytical performance after storing the immunoreagents at 4℃ and 37℃ for 7 days. Little difference was found, indicating satisfactory stability of the method. The present method has been successfully applied to the detection of CA72-4 human serum, and showed a good correlation with the commercially available ELISA kit (r 2=0.9383). This method showed great potential in the fabrication of diagnostic kit for CA72-4, and could be well used in diagnosis of cancer in clinical practice. chemiluminescence enzyme immunoassay, tumor marker, carbohydrate antigen 72-4, human serum

Carbohydrate antigen 72-4 (CA72-4) was proposed by Colcher in 1981. It is a tumor-associated glycoprotein (MW>1000 ku) recognized by monoclonal antibody B72.3 produced by immunizing mice with composition of cancer cell membrane from breast cancer liver metastasis[1,2]. It is found in a high percentage of adenocarcinomas of the stomach, colon, ovary, breast, and lung, and is rarely expressed in most benign or normal adult ― human tissues[3 6]. Generally, the positive rate of CA724 was 49% in the diagnosis of gastric carcinoma, which was higher than that of carcinoembryonic antigen (CEA) and CA19-9 [7] . The combination measurement of www.scichina.com | csb.scichina.com | www.springerlink.com

CA72-4 and CA19-9 shows a high positive rate for mucin cyst diagnosis[7,8]. Spila et al.[9] detected CA72-4 levels of 242 patients who suffered from stomach cancer and found that the positive rate of CA72-4 not only related closely to the differentiation of cancer stage, but also to the development and prognosis of cancer. Moreover, it has been shown that CA72-4 levels connect Received January 18, 2008; accepted May 16, 2008 doi: 10.1007/s11434-008-0428-9 † Corresponding author (email: [email protected]; [email protected]. edu.cn) Supported by the National Basic Research Program of China (Grant No. 2007CB714507) and the National High Technology Research and Development Program of China (Grant No. 2006AA02Z4A8)

Chinese Science Bulletin | October 2008 | vol. 53 | no. 19 | 2958-2963

1 Experimental 1.1 Instrument and reagents BHP9504 microplate chemiluminescence immunoassay reader was from Beijing Hamamatsu Technology Co.

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Ltd., (Beijing China.). DEM-III automatic plate washer was got from Tuopu Analytical Instruments Co. Ltd. Beijing China. DragonMed volume-adjustable pipettes (20―200 μL) were from Dragon Medical Co. Ltd., Shanghai, China. 96-microplate was from Shenzhen Jincanhua Industry Co. Ltd., Shenzhen, China. Electric homoiothermic water bath tank was from Chang’an Scientific Equipment Co. Ltd. Beijing, China. XW-80A blending shaker (Shanghai Jingke) was employed to mix the solutions. CA72-4 antigen was purchased from Fitzgerald (USA). Anti-CA72-4 monoclonal antibody, HRP, and CL substrate solution were obtained from Beijing Chemclin Biotech Co., Ltd. Beijing, China. Human sera from No. 301 Hospital (Beijing, China) were collected. CA72-4 ELISA kits were obtained from DRG (Germany). The quality control sera were obtained from Bio-Rad (USA). Coating buffers were 0.05 mol/L carbonate solution (pH 9.6), 0.05 mol/L phosphate solution (pH 7.4), and 0.06 mol/L citrate solution (pH 4.8). Blocking buffers were imported BSA solution, homemade BSA solution, and casein solution. Washing buffer was 0.05 mol/L phosphate solution with 0.05% Tween-20 (PBST). 1.2 Experimental methods (1) Preparation of solid phase antibody. First, carbonate solution (pH 9.6) was used to dissolve antiCA72-4 antibody. The concentrations of the coating antibody were 1.0, 2.0, 4.0, 6.0, and 7.5 μg/mL, respectively. Afterwards, each well of the microtiter plates was coated with 110 µL antibody solution. The plates were allowed to stand sealed at 4℃ overnight. Then, the plate was washed by PBS solution twice and was gently tapped against tissue paper to remove all fluid. After that, imported BSA solution was added into each well and the plate stood sealed at room temperature for 3 h in order to block the active sites on the plate. Subsequently, the solution in the well was aspirated and the plate was made dry. Finally, the plate was vacuumized and stored at 4℃ for further use. (2) Preparation of standard sample. The serials calibrators were prepared by adding appropriate amount of CA72-4 antigen to the bovine serum and assigned to 0(S0), 2(S1), 6(S2), 20(S3), 60(S4), and 200(S5) U/mL, respectively. The calibrators were stored at 4℃ for further use.

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highly with the tumor size and lymph node involvement, and can be used to indicate the remanet situation of cancer cell after operation[10,11]. Meanwhile, an analytical method of high sensitivity and easy operating is urgently needed to monitor CA72-4 in human serum, which will contribute in large part to the diagnostics, treatment, and prognosis monitoring of a cancer, and exhibit great potential in the clinical application. Being an important sort of analytical method for clinical diagnosis, immunoassay has been widely used to detect tumor markers[12]. At present, immunoassay for the determination of CA72-4 is mainly radioimmunoas― say (RIA)[2,13 15] and enzyme-linked immunoadsorbent assay (ELISA)[16,17]. The disadvantages in using the RIA ― methods[2,13 15], however, are that it requires special disposal and demonstrates a stability problem and long detection period. On the other hand, ELISA methods[16,17] show low detection limit (0.31 U/mL), narrow linear range (0―100 U/mL), and long conduction time. Consequently, they are exempted from popularly employing. Nowadays, chemiluminescence immunoassay (CLIA) has been adopted and applied in routine clinical analysis ― and biomedical researches[18 21] because it does not create radioactive wastes and the instrumentation is simple and is easy to become automatic, and in addition, it shows high sensitivity in determination of tumor mark― ― ers[18 20,22 26]. These reported CLIA methods mainly include magnetic particle-based CLEIA[18,19], immuno― sensors[22 24], and automatic analyzers[25,26]. The fabrication and presentation of a microplate CLEIA for tumor markers, however, have been rarely reported[20]. Microplate CLEIA has shown the merit in the high sensitivity and stability in the detection of tumor markers, and meanwhile, is experiencing rapid popularization. The present work established a microplate CLEIA with high sensitivity and specificity for the determination of CA72-4 in human serum. Results showed that the present method is stable and credible. It could be well suited in the fabrication of diagnostic kits which could be used in the clinical diagnosis, and the facilitation of indigenization of diagnostic reagents.

(3) Preparation of anti-CA72-4-HRP conjugate. The preparation of anti-CA72-4-HRP conjugate was accomplished through periodate oxidation method. The obtained conjugate combines the immunoactivity of the antibody and catalytic activity of HRP. First, 5.0 mg HRP was dissolved in 0.5 mL of deionized water. Then freshly prepared 0.5 mL 0.1 mol/L sodium periodate was added, and the solution was mixed well and was kept stood at 4℃ for 30 min. Then 0.5 mL 0.16 mol/L glycol solution was added to cease the oxidation reaction. After that, 3.0 mg CA72-4 antibody dissolved in 0.5 mL was added to the HRP solution and the resulting mixture was dialysed against carbonate solution (pH 9.5) over night at 4℃. Then equal volume of freshly prepared saturated ammonium sulfate solution was added and the mixture obtained was placed at 4℃ over a period of 1 h. Afterwards, the solution was dialysed against phosphate buffer (pH 7.4) to remove the unreacted materials. Finally, the solution was centrifugated for 30 min to remove the precipitate. The supernatant obtained was mixed with equal volume of glycerol and was stored at −20℃. (4) Immunoassay procedure. Sandwiched immunoreaction was used. First, 25 μL CA72-4 standards or serum samples were added into each well of the test plates. Then, added 100 μL diluted anti-CA72-4-HRP conjugate and incubated for 60 min at room temperature. After the sandwich reaction, washing was performed five times. After the fluid was discarded, 100 μL CL substrate solution was added, and then incubated for 10 min at room temperature (in the dark) and the relative light unit (RLU) was measured. (5) Evaluation of the methodology. Methodological parameters were evaluated as follows: (i) Detection limit: The minimal detected concentration of CA72-4 was ascertained by determining the variability of S0 in 10 replicates. The minimal detected concentration (mean+2SD of zero calibrator) can be calculated from the linear equation. (ii) Precision: Quality control sera in high and low concentration ranges were measured ten times within one assay to obtain the intra-assay precision. Inter-assay precision was calculated by measuring the sera assays three times. (iii) Recovery: CA72-4 with the known concentration of 2.0, 20, and 200 U/mL was added to human serum, and the samples were analyzed by the proposed method 5 times. (iv) Specificity: The cross-reactivity (CR) of CA72-4 was evaluated using 2960

several common tumor markers (AFP, CEA, CA24-2, CA19-9, CA15-3) being 10―100 times higher than their normal concentrations in human body. (v) Stability: For the stability investigation, the components of a kit, including solid phase antibody, anti-CA72-4-HRP conjugate, calibrators, and chemiluminescent substrate, were stored at 4℃ and 37℃ for 3 and 7 days, respectively. After that, they were used to perform the assay. (vi) Clinical coincidence rate:CA72-4 in serum samples was determined with the proposed micro-plate CLEIA and compared with that obtained by a commercial ELISA kit, and the correlation was evaluated. (6) Data analysis. Standards and samples were measured in double wells, and CL intensity values were integrated. Standard curves with linearity were obtained by plotting the logarithm of RLU (logY) against the logarithm of analyte concentration (logX). 1.3 Samples Human sera from No. 301 Hospital were collected. After vein blood sampling, the blood was centrifugated. Then sera were aspirated, subpackaged, and stored at −20℃ for further use.

2 Results and discussion 2.1 Calibrator matrix optimization We investigated the performance difference of several sera when they were used as matrix for calibrator preparation. Three sera, including bovine serum, equine serum, and human serum, were selected. Results showed that human serum and equine serum demonstrated low RLU and high CV, suggesting instability of the calibrators. Bovine serum exhibited that RLU lies in the normal range of the chemiluminescence reader. Meanwhile, a good correlation coefficient (r > 0.9900) was obtained when using bovine serum. Therefore, bovine serum was selected to dilute the stock solution of CA72-4 to prepare the serial calibrators. 2.2 Influence of coating buffer and blocking buffer The composition and concentration of coating buffer and blocking buffer were important factors influencing the sensitivity and linear range of a CLEIA. Therefore, the effects of different coating buffers and blocking buffers on RLUS1/RLUS0, RLUS5/RLUS0, and r, which indicates the sensitivity, linear range, and linearity of the assay, respectively, were studied. As seen in Table 1, coating

JIN Hui et al. Chinese Science Bulletin | October 2008 | vol. 53 | no. 19 | 2958-2963

2.3 Influence of diluent for anti-CA72-4-HRP conjugate and its dilution ratio Anti-CA72-4-HRP conjugate was diluted into a ratio of 1:2000 by homemade diluent I, homemade diluent II, and imported diluent, respectively. After that, the diluted solutions were stored at 4℃ and 37℃ for 3 and 7 days. Then the linear equations were given and CV of r was calculated. Results showed that homemade diluent I gave the lowest CV, indicating the best stability of the assay system. Consequently, anti-CEA-HRP conjugate was serially diluted by homemade diluent I to the ratio of 1:2000, 1:4000, 1:5000, and 1:10000, respectively, and the corresponding RLUS1/RLUS0, RLUS5/RLUS0, and r were obtained and are shown in Table 2. As can be seen, dilution ratio of 1:1000 gave the highest sensitivity, the widest linear range, and the best linearity. Thus dilution ratio of 1:1000 was chosen. 2.4 Influence of incubation conditions The influence of incubation for 30, 45, 60, 75, 90, and 105 min were investigated at 37℃ and room temperature. Results showed that the occurrence of reaction equilibrium between antibodies and antigens is observed within 60 min-incubation at room temperature. Although

2.5 Influence of chemiluminescence reaction time The influence of chemiluminescence reaction time was investigated by testing the RLU of S6 in 5 min interval after adding the chemiluminescent substrate (Figure 1). As can be seen, RLUs increased within 0―10 min, then reached a platform from 10 to 40 min. After 40 min, the RLU decreased. Thus chemiluminescence reaction time of 10 min was selected. 2.6 Evaluation of the methodology (1) Standard curve. Under the optimal reaction conditions, a standard curve was obtained by plotting the logarithm of RLU against the logarithm of analyte concentration with a correlation coefficient of 0.9995, which was suitable for the clinical analysis (Figure 2). (2) Detection limit. The detection limit of 0.18 U/mL was calculated according to the method of 1.2(5). (3) Precision. Two quality control sera of CA72-4 were measured by the proposed method and the results are shown in Table 3. As can be seen, the inter- and intra-coefficients of variation were 5.4% ― 8.3% and 7.8%―9.6%, respectively. (4) Accuracy. CA72-4 with the known concentration of 2.0, 20, 200 U/mL was added to human serum, and the samples were analyzed by the proposed method for 5 times. The average of recovery is shown in Table 4. (5) Specificity. The cross-reactivity (CR) of CA72-4 was calculated by the formula as follows: CR = 100× CCA72-4/Ccross-reactant. Results showed that there was no cross-reactivity with AFP and CEA, and the cross-reactivities of CA24-2, CA19-9, and CA15-3 were all less

Table 1 Influence of different coating buffers and blocking buffers a) (n=3) Coating buffers

Carbonate solution

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37℃ incubation could accelerate the diffusion process, and increase the interaction probability and the reaction speed of the proteins, no obvious difference was observed from that of room temperature incubation. Thus room temperature incubation was finally selected.

Phosphate solution

Blocking buffers

Imported

Homemade

Casein

Imported

Homemade

RLUS0

4976

60533

2539

2140

RLUS1

7707

68409

2944

2979

RLUS5

258628

68413

228987

Citrate solution Casein

Imported

Homemade

Casein

34176

1152

2267

39208

1508

35795

1463

3325

45181

1817

107244

122881

229648

196841

15261

238053

RLUS1/S0

1.55

1.130

1.16

1.39

1.05

1.27

1.47

1.15

1.21

RLUS5/S0

51.98

1.13

90.19

50.11

3.60

199.35

86.83

0.39

157.86

r

0.9985

0

0.9984

0.9771

0.9189

0.9975

0.9984

0.8547

0.9975

a) Experimental conditions: homemade diluent I, dilution ratio 1:2000, 37℃, incubation for 1.5 h, and measurement after adding chemiluminescent substrate for 30 min. JIN Hui et al. Chinese Science Bulletin | October 2008 | vol. 53 | no. 19 | 2958-2963

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buffer of carbonate solution (pH 9.6) and blocking buffer of imported BSA solution correspond to the highest sensitivity, acceptable linear range, and the best linearity. By taking account of the importance of sensitivity and linearity of the proposed method and the practicability in the clinical use, carbonate solution (pH 9.6) and imported BSA solution were selected as the coating buffer and blocking buffer, respectively. In the optimization of antibody coating concentration, 4.0 μg/mL antibody corresponding to the best RLUS1/RLUS0, RLUS5/ RLUS0, and r, was used.

Table 2 Influence of dilution ratio a )(n=3) Dilution ratio

1:1000

1:2000

Table 4 Recovery of the method (n=5)

1:4000

1:5000

1:10000

RLUS0

801

665

537

591

483

RLUS1

2383

1967

1576

893

861

Known (U/mL) 0.66

Added (U/mL)

Tested (U/mL)

Recovery rate(%)

2.00

2.67

101

20.0

20.11

97.2

RLUS5

256349

193018

56729

52317

28736

200

200.1

99.7

RLUS1/S0

2.98

2.96

2.94

1.51

1.78

2.00

22.42

93.0

RLUS5/S0

320.0

290.3

105.6

88.5

59.5

20.0

40.98

102

r

0.9995

0.9245

0.7928

0.5873

0.5427

200

223.16

101

a) Experimental conditions: homemade diluent I, 37℃, incubation for 1.5 h, and measurement after adding chemiluminescent substrate for 30 min.

20.56

Table 5 Stability test results of the analytical system (n=3) Time (d) 3 7

Figure 1 Kinetics curve of chemiluminescence system. Experimental conditions: homemade diluent I, enzyme dilution of ratio 1:1000, and room temperature incubation for 60 min.

Temperature (℃)

S1/S0

r

CV (%)

4

2.48

0.9951

3.6

37

2.53

0.9986

4.2

4

2.57

0.9924

5.1

37

2.50

0.9943

4.8

than 1%, which were absolutely acceptable in the analysis. (6) Stability. Solid phase antibody, anti-CA72-4-HRP conjugate, calibrators, washing buffer, and chemiluminescent substrate were stored at 4℃ and 37℃ for 3 and 7 days, respectively. Then they were used to perform the assay. The results in Table 5 showed that little variation occurred among the different conditions, indicating enough stability of the kit components 2.7 Serum samples analysis CA72-4 in 50 serum samples was determined with the proposed micro-plate CLEIA and compared with that obtained by a commercial ELISA kit. A good correlation was obtained with a satisfied r2 of 0.9383, as shown in Figure 3.

3 Conclusions Through optimizing the conditions of the antibody la-

Figure 2 Standard curve of the proposed method. Table 3 Precision study results

1 Inter-assay (n=10)

2 3

Intra-assay (n=30)

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Low range 3.7―5.6

High range 14.9―22.7

Mean (U/mL)

4.2

18.8

RSD (%)

5.5

6.3

Mean (U/mL)

4.6

17.1

RSD (%)

5.4

8.3

Mean (U/mL)

5.1

18.2

RSD (%)

5.5

6.1

Mean (U/mL)

4.6

18.0

RSD (%)

9.6

7.8

Figure 3 Correlation between a commercial ELISA kit and the proposed CLEIA.

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beling, the parameters of the sandwiched immunoreaction, and the chemiluminescence reaction, a highly sensitive, specific, and simple microplate CLEIA using luminol-H2O2-HRP system was successfully applied to the determination of CA72-4 in human serum. The immunoreagents showed satisfactory stability after being