Determination of tumor marker CA125 by capillary electrophoretic enzyme immunoassay with electrochemical detection

Analytica Chimica Acta 497 (2003) 75–81

Determination of tumor marker CA125 by capillary electrophoretic enzyme immunoassay with electrochemical detection Zhihui He1 , Ning Gao, Wenrui Jin∗ Laboratory of Analytical Science, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Received 24 March 2003; received in revised form 10 June 2003; accepted 11 July 2003

Abstract A novel capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) was developed for a tumor marker cancer antigen 125 (CA125). In this method, after the noncompetitive enzyme immunoreaction, the free enzyme (horseradish peroxidase)-labeled anti-CA125 antibody (Ab∗ ) and the bound enzyme-labeled complex (Ag–Ab∗ ) were separated in a separation capillary and then catalyzed the enzyme substrate (3,3,5,5-tetramethyl-benzidine dihydrochloride, TMB(Red)) and H2 O2 in a reaction capillary following the separation capillary. The product of the enzymatic catalysis reaction (TMB(Ox)) was amperometrically detected on a carbon fiber microdisk bundle electrode. A activity concentration limit of detection (LOD) of 0.29 U/ml, which corresponded to a activity LOD of 1.6 ␮U was achieved. The assay could be used to determine CA125 in human serums from ovary tumor patents. © 2003 Elsevier B.V. All rights reserved. Keywords: Capillary electrophoresis; Electrochemical detection; Immunoassay; Tumor marker

1. Introduction Cancer antigen 125 (CA125), an ovarian cancer associated antigen, is defined by the murine monoclonal antibody OC125, which was obtained by Bast and his colleagues using the ovarian cell line OVCA433 as immunogen [1]. The antigen is located on the surface of ovarian tumor cells, with restricted expression in normal adult tissues such as endocervix, endometrium, tubes, pleura, pericard, peritoneum, and occasional expression in intestine, lung and kidney [2–4]. In sera ∗ Corresponding author. Fax: +86-531-8565167. E-mail address: [email protected] (W. Jin). 1 Present address: Technical Center of Changde Cigarette Factory, Changde 415000, China.

of patients with carcinoma, the CA125 antigen is not exclusive to ovarian carcinoma, but is shown to be elevated in a large number of different cancers [5,6]. The CA125 antigen has also been shown to be present in breast milk (particularly colostrum), ascites, cyst fluid, cervical secretion, uterine secretion and amniotic fluid [7–10]. CA125 is an antigen present on 80% of nonmucinous ovarian carcinomas. It circulates in the serum of patients with ovarian carcinoma and is therefore investigated for possible use as a marker. For healthy human, the concentration levels of CA125 are lower than 35 U/ml [11,12]. Assays for CA125 have great clinical importance. In clinic research, CA125 levels are often measured by immunoradiometric assay [11], enzyme immunoassay [12–15]. These conventional immunoassay have some shortcoming

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such as time-consuming (ca. 4 h), high reagent consumption (ca. 5 ␮l) and complicated operation (over 10 steps). Capillary electrophoresis (CE) is a powerful technique for the separation of macromolecules such as proteins and immunocomplexes [16]. With both superior separation power and high detection sensitivity, CE can separate free antibody or antigen from bound antibody or antigen rapidly, and is especially suitable for immunoassay [17]. The method called capillary electrophoretic immunoassay (CEIA) offers several advantages such as short analysis time (ca. 1.5 h), low reagent consumption (less 1 ␮l), and simple operation (ca. two steps) over conventional immunoassays. The procedure of immunoassay can be simplified by CE separation and many wash steps can be eliminated. In CEIA, UV detection and laser-induced fluorescence (LIF) detection have been used. However, the major disadvantage of the UV detection is the lack of sensitivity. The minimum detectable concentration by UV detection is around 10−6 mol/l. LIF detection is a more general approach to improve sensitivity. Amperometric detection provides excellent sensitivity for the small dimensions associated with CE, while offering a high degree of selectivity towards electroactive species. In our present work, a capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) using a noncompetitive format has been developed and applied to monitor CA125 in serum. In the assay, an excess amount of horseradish peroxidase (HRP)-labeled anti-CA125 antibody, Ab∗ , is added to the sample to form its bound complex, Ag–Ab∗ , with the antigen CA125 (Ag) present in the sample. Then, Ab∗ and Ag–Ab∗ are separated by CE in the separation capillary. Both enter the reaction capillary following the separation capillary and catalyze the reaction of enzyme substrate, reduced form of 3,3,5,5-tetramethyl-benzidine (TMB(Red)) and H2 O2 . The reaction product, oxidized form of 3,3,5,5-tetramethyl-benzidine (TMB(Ox)), is amperometrically detected at a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Since the concentration of TMB(Ox) is much higher than those of the free Ab∗ and the Ag–Ab∗ due to the enzyme amplification, the activity concentration limit of detection (LOD) (3σ) of CA125 is as low as 0.29 U/ml (or a activity LOD of 1.6 × 10−6 U).

The assay was used to determine CA125 in human serum. 2. Experimental 2.1. Apparatus The CE-EIA-ED system used here was the same as in our previous description [18]. Briefly, it consisted of the three parts: a polyacrylamide-coated separation capillary, a polyacrylamide-coated reaction capillary following the separation capillary and a electrochemical detection system. A high-voltage power (Model 9323-HVPS, Beijing Institute of New Technology, Beijing, China) provided a variable voltage of 0–30 kV across the separation capillary (50 ␮m ID, 375 ␮m OD, 15 cm length), with its outlet end at ground potential. The combination of the separation capillary and the reaction capillary (50 ␮m ID, 375 ␮m OD, 5 cm length) was similar to a post-column reactor. The enzyme substrate (TMB(Red)) solution was introduced into the reaction capillary by means of a liquid pressure. The enzyme-catalytic product TMB(Ox) eluting from the reaction capillary was determined by the electrochemical detection system. The reaction capillary and the detection cell were housed in a Faraday cage in order to minimize the interference from noise of external sources. ED at a constant potential was performed with the electrochemical analyzer (Model CHI800, CH Instrument, Austin, TX). ED was carried out with a three-electrode system. It consisted of a carbon fiber microdisk bundle electrode as the working electrode, a saturated calomel electrode (SCE) used as the reference electrode, and a coiled Pt wire (0.3 mm diameter, 5 cm in length) placed at the bottom of the cell as the auxiliary electrode. The carbon fiber microdisk bundle electrodes used here were described previously [19]. Before use, all carbon fiber microdisk bundle electrodes were cleaned in alcohol and washed with double-distilled water for 5 min by an ultrasonicator. 2.2. Capillary treatment The polyacrylamide-coated capillaries were prepared from the uncoated fused-silica capillaries with

Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

50 ␮m ID. The inner surface of capillaries was first pretreated with 1 mol/l NaOH for 30 min and then flushed with water for 30 min. The silane solution adjusted to pH 3.5 by acetic acid containing 0.5% (v/v) ␥-methacryloxypropryltrimethoxysilane (Acros Organics, New Jersey) and 0.5% (v/v) alcohol was sucked up into the capillaries. After reaction proceeded for 1 h at room temperature, the silane solution was removed. Then the capillaries were filled with 3.5% (w/v) deaerated acrylamide solution containing 1 ␮l N,N,N ,N -tetramethylethylenediamine (TEMED) and 2 mg potassium persulphate per milliliter. After 3 h, the excess (not attached) polyacrylamide was sucked away and the capillaries were rinsed with water. After most of the water in the capillaries was removed by aspiration, they were then dried under a N2 stream at 45 ◦ C. 2.3. Immunoassay procedure The immunoassay protocol was a noncompetitive format. A 25 ␮l aliquot of the CA125 standards, or serum samples and a 5 ␮l aliquot of HRP-labeled anti-CA125 antibody were added to a microcentrifuge tube. The solution was incubated for 1 h at room temperature, and then was diluted to 150 ␮l with the running buffer. Then hydrodynamic injection was performed with a 9 cm height for 20 s. A separation high-voltage was applied across the separation capillary and the detection potential was applied at the working electrode. When the separated Ab∗ and Ag–Ab∗ ran from the separation capillary into the reaction capillary, whereupon both catalyzed the reaction of enzyme substrate TMB(Red) and H2 O2 . The reaction product, TMB(Ox), was detected at the outlet of the reaction capillary. In the electrochemical detection, the working microdisk bundle electrode was cemented onto a microscope slide, which was placed over a laboratory-made XYZ micro-manipulator and glued in place in such a way that the microdisk end protruded from the edge of the slide. The position of the microdisk bundle electrode was adjusted (under a microscope) against the outlet of the reaction capillary so that the electrode and the capillary were in contact. This arrangement allowed easy removal and realignment of both the capillary and the electrode. All potentials were measured against SCE. All disposable plastic wares and disposable micro-pipette tips

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used in the assay were autoclaved prior to use in order to denature any contaminants. All solutions were prepared in disposable plastic ware using disposable pipette tips. 2.4. Reagents The CA125 EIA kit (no. 400-10) was purchased from CanAg Diagnostics AB, Gothenburg, Sweden, which consisted of CA125 standards (containing 0, 10, 40, 200 and 500 U/ml), and a solution containing two mouse monoclonal anti-CA125 antibodies (Ov197 and Ov185) (30 ␮g/ml) labeled with HRP. The kit was stored at 4 ◦ C. The ovary cancer serum samples and the results detected by ELISA were provided by Hematological Center, Qilu Hospital, Jinan, China. The serum samples were stored at −20 ◦ C. TMB(Red) (High Pure Grade) was obtained from Amresco Inc. (Solon, OH). A stock standard solution of TMB(Red) (0.020 mol/l) was prepared in double-distilled water and kept in a dark bottle. The running buffer consisted of 2.0 × 10−3 mol/l H2 O2 , 2.5 × 10−4 mol/l Na2 B4 O7 and 9.0 × 10−3 mol/l H3 BO3 (pH 7.4). The substrate solution consisted of 2.0 × 10−4 mol/l TMB(Red), 1.0 × 10−2 mol/l Na2 HPO4 and 5.0 × 10−3 mol/l citric acid (pH 5.0). TMB(Red) or H2 O2 was added to the buffers just before the measurement. The running buffer was renewed every run. All buffers and solutions were stored at 4 ◦ C until use. Unless stated otherwise, all other reagents were of analytical grade and purchased from standard reagent suppliers. All solutions were prepared with double-distilled water. All buffers were filtered through 0.45 ␮m cellulose acetate membrane filters (Shanghai Yadong Resin Co. Ltd., Shanghai, China) before use.

3. Results and discussion 3.1. Optimization of CE-EIA-ED In the assay, the immunoassay protocol is a noncompetitive format. The separated Ab∗ and Ag–Ab∗ catalyze the reaction of the substrates TMB(Red) introduced into the reaction capillary by a liquid pressure and H2 O2 in the running buffer. The catalysis

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reaction is shown as follows [20]:

The enzymatic reaction product TMB(Ox) can be reduced at the carbon fiber microdisk bundle electrode according to the following scheme [20].

Both Ab∗ and Ag–Ab∗ can be detected through measuring TMB(Ox) at the outlet of the reaction capillary. In our experiments, 1.0 × 10−2 mol/l Na2 HPO4 –5.0 × 10−3 mol/l citric acid (pH 5.0) containing 2.0 × 10−4 mol/l TMB(Red) buffer was used as the substrate. To obtain the high and rannow electrophoretic peak of Ab∗ , different running buffers of pH 7.4 (Na2 HPO4 –citric acid, Tris–HCl, Na2 B4 O7 –H3 BO3 ) were tested. It is found that 2.5 × 10−4 mol/l Na2 B4 O7 –9.0 × 10−3 mol/l H3 BO3 of pH 7.4 is better as the running buffer. The migration time, tm , the peak area, q, the width at the half-peak, W1/2 , and the number of theoretical plates, N, of Ab∗ at different concentrations of H2 O2 , CH2 O2 , are listed in Table 1. tm and N are almost constant. When CH2 O2 < 1.0 × 10−3 mol/l, q increases rapidly with increasing CH2 O2 . When CH2 O2 is between 1.0 × 10−3 mol/l and 2.0 × 10−3 mol/l, q is almost a constant, which indicates the saturation of H2 O2 for HRP-labeled. However, when CH2 O2 > 2.0 × 10−3 mol/l, q decreases with increasing CH2 O2 . This is because the activity of HRP is less sensitivity to excess H2 O2 [21]. The maximum q was obtained using 2.0 × 10−3 mol/l H2 O2 . The value was used for determination of CA125. It is possible

that H2 O2 reacts with the capillary and the carbon fiber electrode in a reproducible manner because of good reproducible results. Therefore, H2 O2 did not affect the results, when the product TMB(Ox) was detected. Fig. 1 shows the relationship between q and the applied detection potential, Ed . When Ed is more positive than 0.10 V, q increases with decreasing Ed . When Ed is more negative than 0.10 V, q is almost a Table 1 The values of tm , q, W1/2 and N at different concentrations of H2 O2 in the running buffer, CH2 O2 (running buffer, 2.5×10−4 mol/l Na2 B4 O7 –9.0 × 10−3 mol/l H3 BO3 (pH 7.4); 1.0 ␮g/ml Ab∗ ; substrate solution, 2.0 × 10−4 mol/l TMB(RED) in 1.0 × 10−2 mol/l Na2 HPO4 –5.0 × 10−3 mol/l citric acid (pH 5.0); separation capillary, 25 cm × 50 ␮m ID; reaction capillary, 5 cm × 50 ␮m ID; hydrodynamic injection, 9 cm for 20 s; separation voltage, 20 kV; detection potential, 0.00 V vs. SCE) CH 2 O 2 (10−3 mol/l)

tm (min)

q (nC)

W1/2 (s)

N (104 )

0.2 0.5 1.0 2.0 5.0 10

12.2 12.1 12.0 11.8 11.6 11.5

0.85 1.15 2.35 2.40 1.85 1.03

8.3 8.5 8.6 8.6 8.4 8.2

4.3 4.0 3.9 3.8 3.8 3.9

Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

2.8

q (nC)

2.4 2.0 1.6 1.2 0.8 0.5

0.4

0.3

0.2

0.1

0.0

-0.1

-0.2

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After the noncompetitive reaction was completed, the solution containing Ab∗ and Ag–Ab∗ was injected into the separation capillary and Ab∗ and Ag–Ab∗ were separated by CE in the separation capillary. Both catalyzed TMB(Red) and H2 O2 in the reaction capillary following the separation capillary. The reaction product, TMB(Ox), was detected at the outlet of the reaction capillary on the carbon fiber microdisk bundle electrode. Thus, two peaks corresponding to Ab∗ and Ag–Ab∗ should appear in the electropherograms. The electropherograms obtained are shown in Fig. 2

Ed (V vs SCE) Fig. 1. Relationship between the detected electric charge, q, and the detection potential, Ed ; 2.0×10−3 mol/l H2 O2 and other conditions are same as in Table 1.

constant. Ed of 0.00 V was used because of larger q and lower noise. tm , q, W1/2 , and N at different separation voltages, Vs , are listed in Table 2. From Table 2, it can be seen that tm and W1/2 decrease and N increases with increasing Vs . This is because higher values of Vs give higher electroosmotic flow rate. When Vs < 20 kV, q is a constant because of identical coulometric efficiency. However, when Vs > 20 kV, q reduces with increasing Vs . In this case, tm shortened and only a part of TMB(Ox) can be reduced at the working electrode, i.e. the coulometric efficiency is decreased. Therefore, 20 kV for Vs was chosen in our experiments. 3.2. CE-EIA-ED for CA125 In this method the noncompetitive format was performed. CA125 (Ag) reacted with an excess amount of the solution containing two HRP-labeled monoclonal anti-CA125 antibodies (Ab∗ ) from the EIA kit. Table 2 The values of q, tm , W1/2 and N at different separation voltages, Vs (2.0 × 10−3 mol/l H2 O2 and other conditions are same as in Table 1) Vs (kV)

tm (min)

q (nC)

W1/2 (s)

N (104 )

10 15 18 20 22 25

13.1 12.6 12.2 11.8 11.5 11.1

2.40 2.41 2.36 2.40 1.88 0.90

17.8 10.5 8.8 8.6 8.0 6.9

1.1 2.9 3.8 3.8 4.1 5.2

Fig. 2. Electropherograms of the solutions for different concentrations of CA125. Concentration of CA125 (U/ml): (1) 0; (2) 1.67; (3) 6.67; (4) 33.3; (5) 83.3; 2.0 × 10−3 mol/l H2 O2 and other conditions are same as in Table 1.

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Table 3 Results detected and recovery of CA125 in serum samples (2.0 × 10−3 mol/l H2 O2 and other conditions are same as in Table 1) Sample

Determined value (U/ml)

Average value (U/ml)

Added value (U/ml)

Observed value (U/ml)

Recovery (%)

I

23.5 24.7 25.3

24.5

13.3 20.0 25.0

37.2 43.5 49.8

103 94 98

II

35.3 34.2 34.7

34.7

13.3 25.0 33.3

49.5 58.5 66.8

107 97 96

at different concentrations of CA125. With increasing the concentration of CA125, q of peak 1 decreases and q of peak 2 increases. According to the principle of the noncompetitive assay, peaks 1 and 2 should be the peak of Ab∗ and the peak of Ag–Ab∗ , respectively. Although the post-capillary catalysis reactor and the end-capillary amperometric detector could introduce the post-capillary zone broadening, the enough resolution for the both peaks did not affect their measurement. It can be found from Fig. 2 that the peak 1 (the peak of Ab∗ ) consists of two peaks. The two monoclonal antibodies in the EIA kit should be responsible for that. It was also verified by the fact that peak 2 (the peak of Ag–Ab∗ ) was obviously divided into two peaks, when the concentration of CA125 was increased. Therefore, the total peak area of the peak 2 was used for quantification of CA125. The calibration curve based on the peak of Ag–Ab∗ is shown in Fig. 3. The points represent the average q detected

1.5

q (nC)

1.2 0.9 0.6 0.3 0.0

0

20

40

60

for three runs. LOD for the noncompetitive assay calculated by using the mean q of the Ag–Ab∗ peak for the zero-dose CA125 plus three times its standard deviation calculated from 10 trials was 0.29 U/ml. According to Hagen–Poiseuille equation, the injection volume calculated was 5.5 nl for the hydrodynamic injection with 9 cm height for 20 s. Therefore, a activity LOD of 1.6 × 10−6 U can be obtained. The response for a series of six injections of 6.67 U/ml CA125 resulted in a relative standard deviation of 4.8% for tm and 3.9% for q. In order to verify the method, two serum samples from different ovary tumor patients were detected according to the procedure described in the experimental section. The determined results of the diluted samples are shown in Table 3. The concentrations of CA125 in the two samples obtained by the calibration curve are 139 and 201 U/ml, respectively. Also, the two serum samples were determined by Hematological Center, Qilu Hospital (Jinan, China) using the routine ELISA for comparison. The values of CA125 were found to be 150 and 209 U/ml, respectively, by ELISA. In order to prove the reliability of the method, a certain amount of standard CA125 was added to the two serum samples. Then, the serum samples with the standard CA125 were measured. Thus, we can obtain the recovery. The results are listed in Table 3. The determined recovery of the method was between 94 and 107%. Obviously, the present CE-EIA-ED is a simpler and more time-saving.

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CCA125 (U/mL) Fig. 3. Calibration curve based on the total peak area of the complex of CA125 with its antibody, Ag–Ab∗ . Running buffer, 2.5 × 10−4 mol/l Na2 B4 O7 –9.0 × 10−3 mol/l H3 BO3 (pH 7.4) containing 2.0 × 10−3 mol/l H2 O2 and other conditions are same as in Table 1.

4. Conclusion The developed CE-EIA-ED of CA125 with the noncompetitive format is a new useful method with high selectivity, low LOD and low sample consumption. It should be noted that this approach is not limited to

Z. He et al. / Analytica Chimica Acta 497 (2003) 75–81

the determination of CA125. In many commercially available enzyme immunoassay kits, HRP is labeled on antigen or antibody, and the TMB(Red) is used as enzyme substrate. Therefore, CE-EIA-ED with a noncompetitive format based on the catalysis action of HRP can easily be used to determine other antigens or antibodies. This method is useful where the HRP enzyme label is available and a fluorescent label is not. We think that CE-EIA-ED will become a useful tool in immunological assays. Acknowledgements This project was supported by the National Natural Science Foundation of China (no. 20235010) and the State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. References [1] R.C. Bast Jr., M. Feeney, H. Lazarus, L.M. Nadler, R.B. Colvin, R.C. Knapp, J. Clin. Invest. 68 (1981) 1331. [2] R.C. Bast Jr., T.L. Klug, E.St. John, E. Jenison, J.M. Niloff, H. Lazarus, R.S. Berkowitz, T. Leavitt, C.T. Griffiths, L. Parker, V.R. Zurawski Jr., R.C. Knapp, N. Engl. J. Med. 309 (1983) 883. [3] E.J. Nouwen, P.G. Hendrix, S. Dauwe, M.W. Eerdekens, M.E. De Broe, Am. J. Pathol. 126 (1987) 230.

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