Enhancement of the Sensitivity of a Capillary Electrophoresis

Anal. Chem. 2001, 73, 5616-5619

Enhancement of the Sensitivity of a Capillary Electrophoresis Immunoassay for Estradiol with Laser-induced Fluorescence Based on a Fluorescein-labeled Secondary Antibody Yong-Cheng Wang, Ping Su, Xin-Xiang Zhang,* and Wen-Bao Chang

Department of Chemical Biology, College of Chemistry, Peking University, Beijing, 100871, China

A competitive immunoassay for estradiol (E2) based on capillary electrophoresis was established. This method was based on the competitive reaction of complete antigen and E2 with a limited amount of monoclonal antibody, with fluorescein isothiocyanate (FITC)-labeled secondary antibody as a fluorescence probe. The addition of the thermally reversible hydrogel, poly-N-isopropylacrylamide (pNIPA) in the buffer as a replaceable packing material improved reproducibility and resolution of the method. This capillary electrophoresis immunoassay with laserinduced fluorescence can be applied to determine E2 with good precision at concentrations as low as 9 pg/mL. Details of typical separations of immunological complex and free reactants are presented. Sex hormones play essential roles in the development and differentiation of organs, signal transduction control and metabolism adjustment. Assessment of estradiol (E2) concentration in serum, plasma, saliva, and hair is of great value for endocrinological investigation. Clinical monitoring of estrogens in circulation is desirable for the treatment and study of hormone-dependent diseases,1,2 monitoring estrogen supplementation,3-4 determining functional infertility,5 and predicting ovulation.6 However, the extreme fluctuations in the circulating levels of E2 make measurement difficult. Serum E2 levels vary over a range from 150 to 1000 pmol/L during the menstrual cycle in premenopausal women, and the E2 levels decrease to about 15-51 pmol/L7,8 after menopause. Different methods, such as HPLC, immunoassay and GC/MS have been applied in the measurement of E29-10. Immunoassay is the * To whom correspondence should be addressed. Fax: +8610-62751708. E-mail: [email protected]. (1) Sittisomwong, T.; Suneja, A.; Kudelka, A. P.; Verschraegen, C. F.; Kavanagh, J. J. Eur. J. Gynaecol. Oncol. 2000, 21, 348-354. (2) Chan, K. C.; Muschik, G. M.; Issaq, H. J.; Siiteri, P. K. J. Chromtogr. A 1995, 690, 149-154. (3) Tatar, S.; Atmaca, S. Pharmazie 1996, 51, 251-252. (4) Prestwood, K. M.; Kenny, A. M.; Unson, C.; Kulldorff, M. J. Clin. Endocrinol. Metab. 2000, 85, 4462-4469. (5) Haning, R. V.; Levin, R. M.; Behrman, H. R.; Kase, N. G.; Speroff, L. J. Obstet. Gynaecol. 1979, 54, 442-447. (6) Roger, M.; Grenier, J.; Houlbert, C.; Castanier, M.; Feinstein, M. C.; Scholler, R. J. Steroid Biochem. 1980, 12, 403-410. (7) Reed, M. J.; Cheng, R. W.; Noel, C. T.; Dubley, A. F.; James, H. T. Cancer Res. 1983, 43, 3940-3943. (8) Jensen, J.; Riis, B. J.; Hummer, L.; Christiansen, C. Br. J. Obst. Gynaecol. 1985, 92, 260-265.

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preferred technique because of its specificity and sensitivity. Radioimmunoassay (RIA),11-13 enzyme immunoassay (EIA),14 chemiluminescence immunoassay (CLIA),15 and time-resolved immunoassay (TrFIA)16,17 are widely used in screening and determination of E2. However, some immunoassays are not sensitive enough for E2 determination, and others have high sensitivity but are time-consuming. A quick and sensitive immunoassay for measuring E2 levels in biological samples would have practical application. Capillary electrophoresis combined with immunoassay has been demonstrated to be a useful technique for the separation and analysis of biological compounds.18-21 This technique allows rapid analysis, high sensitivity, small sample consumption, and easy automation. Nevertheless, proteins in the sample may be absorbed onto the capillary surface, which changes the composition and the charges of the capillary wall and detrimentally affects resolution and reproducibility.22-23 There are usually two ways to lessen the binding of proteins to the capillary wall surface. One approach is to modify the capillary wall with polymers, which can decrease the interaction between the protein and the inner-wall of capillary.24-25 However, the polymer-coating process is timeconsuming, and the modified capillary has a limited life. The other (9) Busico, F.; Moretti, G.; Cartoni, G. P.; Rosati, F. J. High Resolt. Chromatogr. 1992, 15, 94-97. (10) Bayoumi, A. EI.; Gendy, A. E. EI.; Ragehy, N. A.; Kawy, M. Bull. Fac. Pharm. 1993, 31, 303-305. (11) Mertens, R.; Liedtke, R. J.; Batjer, J. D. Clin Chem. 1983, 29, 1961-1963. (12) Schioeler, V.; Thode, J. Clin. Chem. 1988, 34, 949-952. (13) Worthman, C. M.; Stallings, J. F.; Hofman, L. F. Clin. Chem. 1990, 36, 1769-1773. (14) Pandey, P. K.; Shrivastav, T. G.; Kumari, G. L.; Rao, P. N.; Grover, P. K.; Murthy, H. G. K. Clin. Chim. Acta 1990, 190, 175-184. (15) Rodriguez-Espinosa, J.; Otal-Entraigas, C.; Gascon-Roche, N.; Mora-Brugues, J.; Urgell-Rull, E.; Bordas-Serrat, J. R.; Viscasillas-Molins, P. Clin. Chem. Lab. Med. 1998, 38, 969-974. (16) Thomas, C. M. G.; Van-den-Berg, R. J.; Segers, M. F. G.; Bartelink, M. L.; Thien, T. Clin. Chem. 1993, 39, 2341-2342. (17) Tang, D.; Wang, Y. C.; Chang, W. B.; Ci, Y. X. Chin. J. Anal. Chem. (in Chinese) 1999, 27, 899-903. (18) Schmalzing, D.; Nashabeh, W. Electrophoresis 1997, 18, 2184-2193. (19) Lam, M. T.; Wan, Q. H.; Boulet, C. A.; Le, X. C. J. Chromatogr. A 1999, 853, 545-553. (20) Caslavska, J.; Allemann, D.; Thormann, W. J. Chromatogr. A 1999, 838, 197-211. (21) Choi, J.; Kim, C.; Choi, M. J. Anal. Biochem. 1999, 274, 118-124. (22) Li, J.; Fritz, J. S. J. Chromatogr. A 1999, 840, 269-279. (23) Graul, T. W.; Schlenoff, J. B. Anal. Chem. 1999, 71, 4007-4013. (24) Huang, M. X.; Lee, M. L. J. Microcolumn Sep. 1992, 4, 491-496. 10.1021/ac010537a CCC: $20.00

© 2001 American Chemical Society Published on Web 10/06/2001

choice is to modify the background electrolyte. It has been previously demonstrated that thermosensitive poly-N-isopropylacrylamide (pNIPA), a polymer, can be used to control electroosmotic flow.26 At the lower critical temperature of about 32 °C,27 pNIPA can reversibly form and cleave hydrogen bonds. The hydrogen bond response of pNIPA above and below a critical temperature is believed to change the surface of the polymer from being hydrophilic to being hydrophobic nature. This eliminates the adsorption of proteins onto the surface.28 The minor adsorption and improvement of background electrolyte make it possible for separation and analysis with high resolution and reproducibility. Although the resolution of E2 analysis using capillary electrophoresis was satisfactory,29 much effort was devoted to improving the sensitivity of the assay. An amplification system using labeled secondary antibodies was a promising technique for improving the sensitivity of immunoassay.30-31 The reactive activity of a secondary antibody cannot be significantly influenced by the binding of small probe molecule. Meanwhile, the secondary antibody can be multivalently bound to a primary antibody to form a constant immunological complex. In this research, fluorescein-labeled secondary antibody was utilized with CE-LIF for the determination of E2. A thermally reversible hydrogel pNIPA was used as a dynamic modifier in the capillary electrophoresis. A capillary electrophoretic immunoassay of free E2 was then established. This immunoassay was based on the competitive reaction of antigen and free E2 with a limited amount of monoclonal antibody, with FITC-labeled secondary antibody as a tracer. Both capillary zone electrophoresis and hydrogel-modified capillary electrophoresis were valued for suitability for free E2 determination. The employment of hydrogel enhanced the separation, and the fluoresceinated secondary antibody effectively improved the sensitivity of analysis. This method has a good potential in the determination of E2 in biological fluids and environmental samples.

supplied by Zhaohui Pharmaceutics (Beijing, China). Fluorescein isothiocyanate-labeled secondary antibody (Ab2*) was purchased from Huamei Biochemicals (Beijing, China). N-isopropylacrylamide was synthesized in our laboratory. Running buffer containing 100 mmol/L Tris borate (pH 8.0), 0.5 mol/L sodium carbonate solution (pH 9.5), and other solutions were prepared by dissolving the reagents in pure water supplied by a Milli-Q Plus water purification system (Millipore, Bedford, MA). The antigen (E2BSA) was synthesized by coupling E2 with BSA (Mr, 68 000), and the monoclonal antibody (Ab) was prepared and purified from ascites collected from immunized mice (Monoclonal Laboratories, Department of Biology, Peking University, China). Preparation of Calibration Curve. Into nine identical samples containing 49.4 ng/mL antigen, 0.6 µg/mL monoclonal antibody, and 2.1 µg/mL labeled secondary antibody were added different concentrations of estradiol. Final concentrations of estradiol were 0, 20, 25, 30, 50, 200, 600, 1500, and 5000 pg/mL. Each sample was determined 5 times at the same concentration. Procedures of Capillary Electrophoretic Immunoassay (CEIA). The poly-N-isopropylacylamide (pNIPA) was synthesized according to our previous work.30 A fixed amount of pNIPA was added to Tris borate buffer of a suitable pH and concentration, which was subsequently packed into an uncoated capillary. The mixture of E2, E2-BSA, Ab, and Ab2* was injected into the positive end with the pressure at 20 psi for 5 s, and then it was separated in temperature control mode. The concentrations of Ab2*, E2Ab-Ab2*, and Ag-Ab-Ab2* were calculated by peak areas, and the concentration of E2 could be calculated. Immunoassay Principle of Secondary Antibody System. Competitive and noncompetitive models are basic analytical patterns in the immunoassay. In the competitive model, at least a kind of analytical reagent is limited. In our experiment, estradiol was determined by competitive immunoassay with labeled secondary antibody. E2-BSA (Ag) and estradiol (E2) were added to the limited monoclonal antibody (Ab) solution and incubated for 30 min at 37 °C. Then labeled secondary antibody(Ab2*) was added to the above solution and incubated for 60 min at 37 °C. The whole procedure is illustrated in the following equations:

MATERIALS AND METHODS Apparatus. The experiments were performed using a capillary electrophoresis system of the P/ACE 5000 series equipped with a laser-induced fluorescence (LIF) detector obtained from Beckman-Coulter Instruments (Fullerton, CA). A 488-nm argon ion laser provided fluorescence excitation. The fluorescence intensity was measured after passing through a 488-nm cutoff and a 520nm band-pass filter. Material and Reagents. The fused-silica capillary (75-µm i.d., 375-µm o.d.) was supplied by Yongnian Fiber Company (Hebei, China). The capillary, 57 cm in total length with 50 cm to the detection window, was employed for the separation. E2 and bovine serum album (BSA) were obtained from Sigma (St. Louis, MO). 2-Propanol, acrylonitrile, ammonium persulfate (APS), and N,N,N′N′tetramethylethylenediamine (TEMED) were purchased from Beijing Chemicals (Beijing, China). Fluorescein isothiocyanate was

Ag + E2 + Ab(limited) f Ag-Ab + E2-Ab + Ag + E2 (1)

(25) Makino, K.; Suzuki, K.; Sakurai, Y.; Okano, T.; Ohshima, H. Colloids Surf. A 1995, 103, 221-226. (26) Haruma, K.; Keiji, F.; Yoshiro, M. Colloid Polym. Sci. 1992, 270, 53-57. (27) Zhang, X. X.; Li, J.; Gao, J.; Sun, L.; Chang, W. B. Electrophoresis 1999, 20, 1998-2002. (28) Hirokawa, Y.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379-6380. (29) Luppa, P.; Hauck, S.; Schwab, I.; Birkmayer, C.; Hauptmann, H. Clin. Chem. 1995, 41, 564-570. (30) Su, P.; Wang, Y. C.; Zhang, X. X.; Sun, L.; Chang, W. B. Anal. Chim. Acta 2000, 218, 137-143. (31) Katoh, S.; Kobe, T. J. Chromatogr. A 1999, 852, 97-104.

Ag-Ab + E2-Ab + Ag + E2 + Ab2* f Ag-Ab-Ab2* + E2-Ab-Ab2* + Ab2* + Ag + E2 (2) RESULTS AND DISCUSSION On the assumption that some parameters, such as column length and temperature were held constant, parameters affecting resolution and speed of separation, including (i) ionic strength of the buffer, (ii) concentration of hydrogel in the buffer, and (iii) applied voltage were studied. Separation conditions were optimized for the study in this immunoassay. Effect of Incubation Time. The type of separation was a hydrogel capillary electrophoresis in which labeled secondary antibody (Ab2*) and monoclonal antibody(Ab) were added to a sample. In this experiment, five identical samples containing 0.6 µg/mL of antibody and 2.1 µg/mL of labeled secondary antibody were incubated for different lengths of time. The incubation time had a great influence on the binding rate of Ab2* to Ab. Times of 10∼90 min were studied (Figure 1). When the incubation time is 10 min, the binding rate is 15%. Increasing the incubation time increased the binding rate. When the incubation time was prolonged to 60 min, the binding rate reached 65%. The binding Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 1. Effect of incubation time: 100 mmol/L Tris borate buffer, pH 8.0; applied voltage, 25kV; capillary temperature, 25 °C; others as described in text.

rate was constant, even when the incubation time was over 60 min. The results showed the best incubating time for Ab2* and Ab was 60 min. Affinity Constant in Solution of Ab-Ab2 Complexes. During analysis in the capillary electrophoresis immunoassay, the stability of the Ab-Ab2 complex must be maintained. This stability of the complex was characterized by the affinity constant in solution, which was measured by enzyme-linked immunosorbent assay (ELISA). In this experiment, monoclonal antibody (Ab) at various concentrations (3.71 × 10-10 to 1.90 × 10-7 mol/L) was first incubated in 0.01 mol/L PBS (pH 7.4) with HRP-labeled secondary antibody (Ab2*) at a constant concentration (0.4 µg/ mL) at 37 °C. After the equilibrium was reached, 100 µL of each mixture was transferred into the wells of microtitration plate coated with Ab (100 µL per well, at 8 µg/mL in 0.05 mol/L pH 9.6 sodium carbonate, overnight at 4 °C), followed by an incubation for 1 h at 37 °C. After the washing procedure and an addition of enzyme substrate, the measured optical density determined the bound Ab2-HRP, which subsequently quantified the concentration of free Ab2-HRP. Then the affinity constant was calculated according to the Scatchard equation presented by Friguet et al.32 The affinity constant in solution was 3.43 × 107 L/mol, which ensured the Ab-Ab2* to be consistent in the capillary electrophoresis immunoassay. Effect of the pH and the Concentration of the Buffer. The effect of the different buffer ionic strengths on resolution was studied by CZE. For a given set of conditions, the concentration of the buffer has important effects on resolution (Rs) and migration time (tm). When the buffer concentration increased, tm and Rs for EOF increased. The buffer containing 100 mmol/L Tris-borate was selected for immunological reaction. It is important, for the sake of resolution, time of experiment, and Joule heating, to pay attention to the ionic strength. The pH of the buffer has an important effect on the surface characteristics of the fused-silica capillary and the effective electric charge of the ion. Because the hydrogel polymers swelled in aqueous solution and allowed permeation of different-molecularweight compounds, an equilibrated state was attained between the hydrogel polymer layers and the buffer solution, which eliminated the interactions between proteins and the capillary surface and allowed highly efficient and reproducible capillary electrophoretic separations of proteins. The pH of the buffer has a large effect on the conformation of antigen and antibody. (32) Friguet, B.; Chaffotte, A. F.; Djavadi-Ohaniance, L.; Goldberg, M. E. J. Immunol. Methods 1985, 77, 305-319.

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Figure 2. Separation of bound secondary antibody and free secondary antibody using different concentrations of hydrogel: 1, Ab2*-Ab; 2, Ab2*; (A) 0.27 mg/mL; (B) 0.53 mg/mL; (C) 0.79 mg/ mL, 1.25 mg/mL; others as in Figure 1.

Effect of Hydrogel Concentration. With many hydrophobic groups in their structures, Ab2* and its complex would be adsorbed to the inner wall of the capillary, which would cause fluctuation of the migration time. Hydrogel was added in our study. Figure 2 shows that resolution and migration time increased as the gel concentration increased. The results (Figure 2) show that resolution of Ab2* and the Ab2*-Ab compound was well-improved. The hydrogel we used was poly-N-isopropylacrylamide, which could form a linear hydrophobic internal space under certain conditions and decrease the interaction of Ab2*and its complex with the inner wall. With the addition of hydrogel, an uncoated fused-silica capillary can be applied very conveniently in the separation of the immunoassay system. Optimization of Immunological Reactions. Optimization of immunological reactions was performed. In this experiment, a mixture with Ab2*, E2-Ab-Ab2*, Ag-Ab-Ab2*, Ag, and E2 was injected into a capillary. The optimized concentrations of antigen, monoclonal antibody, and labeled secondary antibody determined in solution were 49.4 ng/mL, 0.6 µg/mL, and 2.1 µg/mL, respectively. Immunological Reaction. In our experiments, capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) using a replaceable hydrogel was demonstrated to be a useful technique for performing the separation step required in an immunoassay of estradiol. This type of immunoassay is a competitive assay in which antigen (E2-BSA), monoclonal antibody, and labeled secondary antibody are added to a sample. Analyte, in this case, unlabeled hapten (estradiol), of different concentrations competes with the antibody to form immunological complexes. The ratios of bound and free FITC-Ab2 changed according to different estradiol concentrations in the samples (Figure 3), and the changes in area ratios of two peaks were consistent with estradiol concentrations. The calibration curve (Figure 4) for estradiol can be acquired by plotting the relative area over the maximum area ratio (the area ratio of bound to free tracer at zero concentration of estradiol) against the log concentrations of

Figure 4. Calibration curve for standard estradiol using 1.25 mg/ mL hydrogel. Separation conditions as in Figure 1.

Figure 3. CE-LIF immunoassay of estradiol using 1.25 mg/mL hydrogel: 1, Ab2*-Ab-g; 2, Ab2*-Ab-E2; 3, Ab2*; (A) 0.1, ng/mL; (B) 0.6, ng/mL; (C) 3.0, ng/mL; others as in Figure 1.

estradiol. The linear range (n ) 8, r ) 0.995) and detection limit for estradiol were ∼25 pg/mL to 5ng/mL and 9.0 pg/mL, respectively. Because the analogues of estradiol would interfere with the determination of estradiol in some methods, it is necessary to estimate the interferences of them in the study. The possibility of interference with the immunological process from analogues can be estimated from the cross-reaction. The cross-reactions of analogues of estradiol, determined by ELISA, are estrone, 2.4%; estriol, 4.7%; and progesterone, 1.3%. The results show that there was no interference from those analogues in the determination of estradiol at the applied concentration level. Analytical Application. Simulated samples were analyzed by this method. Two samples, one with low concentration (150 pg/ mL) and the other with high concentration (1.0 ng/mL) of estradiol, were diluted by a de-hormoned serum from healthy volunteers. Seven repetitive experiments were made under the same conditions. The analytical results are listed in Table 1. Results show that this method could be applied satisfactorily as a reliable means for detecting estradiol in serum. CONCLUSIONS With the thermal reversible hydrogel and labeled secondary antibody, a simple and rapid capillary electrophoretic immunoassay of estradiol was demonstrated in an uncoated fused-silica (33) Worthman, C. M.; Stallings, J. F.; Hofman, L. F. Clin. Chem. 1990, 36, 1769-1773.

Table 1. Recovery of Samples Using 1.25 mg/mL Hydrogel Capillary Electrophoresis sample content (ng/mL)

added (ng/mL)

detection (ng/mL, n ) 7)

RSD (n ) 7)

recovery (%, n ) 7)

0 0

0.15 1.0

0.138 0.923

2.57 2.16

92.0 92.3

capillary. Adding hydrogel into the buffer can minimize the adsorption of antibody, antigen, secondary antibody, and their complexes to the inner wall of capillary. These results have demonstrated that secondary antibody can be applied to separate estradiol rapidly and efficiently in the capillary packed with replaceable poly-N-isopropylacrylamide (pNIPA) hydrogel. This method can be employed to independently detect estradiol in serum at any time. Even though this technique’s detection limit (about 10-11 mol/L) has not achieved RIA’s level (about 10-12 mol/L33), it is possible to apply this method in some clinic analyses when the concentration of estradiol is abnormally high. ACKNOWLEDGMENT The authors acknowledge financial support from the National Natural Science Foundation of China (20075003, 20075001) and Excellent Young Teacher Program from MOE, P.R.C. We also thank Dr. Peter Osborne for his helpful discussion.

Received for review May 9, 2001. Accepted August 29, 2001. AC010537A

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