Enhancement of the Sensitivity of a CapillaryElectrophoresis Immunoassay for Estradiol withLaser-induced Fluorescence Based on aFluorescein-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 oncapillary electrophoresis was established. This methodwas based on the competitive reaction of complete antigenand E2 with a limited amount of monoclonal antibody,with fluorescein isothiocyanate (FITC)-labeled secondaryantibody as a fluorescence probe. The addition of thethermally reversible hydrogel, poly-N-isopropylacrylamide(pNIPA) in the buffer as a replaceable packing materialimproved reproducibility and resolution of the method.This capillary electrophoresis immunoassay with laser-induced fluorescence can be applied to determine E2 withgood precision at concentrations as low as 9 pg/mL.Details of typical separations of immunological complexand free reactants are presented.

Sex hormones play essential roles in the development anddifferentiation of organs, signal transduction control and metabo-lism adjustment. Assessment of estradiol (E2) concentration inserum, plasma, saliva, and hair is of great value for endocrinologi-cal investigation. Clinical monitoring of estrogens in circulationis desirable for the treatment and study of hormone-dependentdiseases,1,2 monitoring estrogen supplementation,3-4 determiningfunctional infertility,5 and predicting ovulation.6 However, theextreme fluctuations in the circulating levels of E2 make measure-ment difficult. Serum E2 levels vary over a range from 150 to 1000pmol/L during the menstrual cycle in premenopausal women, andthe E2 levels decrease to about 15-51 pmol/L7,8 after menopause.Different methods, such as HPLC, immunoassay and GC/MS havebeen applied in the measurement of E29-10. Immunoassay is the

preferred technique because of its specificity and sensitivity.Radioimmunoassay (RIA),11-13 enzyme immunoassay (EIA),14

chemiluminescence immunoassay (CLIA),15 and time-resolvedimmunoassay (TrFIA)16,17 are widely used in screening anddetermination of E2. However, some immunoassays are notsensitive enough for E2 determination, and others have highsensitivity but are time-consuming. A quick and sensitive immu-noassay for measuring E2 levels in biological samples would havepractical application.

Capillary electrophoresis combined with immunoassay hasbeen demonstrated to be a useful technique for the separationand analysis of biological compounds.18-21 This technique allowsrapid analysis, high sensitivity, small sample consumption, andeasy automation. Nevertheless, proteins in the sample may beabsorbed onto the capillary surface, which changes the composi-tion and the charges of the capillary wall and detrimentally affectsresolution and reproducibility.22-23 There are usually two ways tolessen the binding of proteins to the capillary wall surface. Oneapproach is to modify the capillary wall with polymers, which candecrease the interaction between the protein and the inner-wallof capillary.24-25 However, the polymer-coating process is time-consuming, and the modified capillary has a limited life. The other

* To whom correspondence should be addressed. Fax: +8610-62751708.E-mail: zxx@pku.edu.cn.(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.

(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. (inChinese) 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.

Anal. Chem. 2001, 73, 5616-5619

5616 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001 10.1021/ac010537a CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 10/06/2001

choice is to modify the background electrolyte. It has beenpreviously demonstrated that thermosensitive poly-N-isopropyl-acrylamide (pNIPA), a polymer, can be used to control electro-osmotic flow.26 At the lower critical temperature of about 32 °C,27

pNIPA can reversibly form and cleave hydrogen bonds. Thehydrogen bond response of pNIPA above and below a criticaltemperature is believed to change the surface of the polymer frombeing hydrophilic to being hydrophobic nature. This eliminatesthe adsorption of proteins onto the surface.28 The minor adsorptionand improvement of background electrolyte make it possible forseparation and analysis with high resolution and reproducibility.

Although the resolution of E2 analysis using capillary electro-phoresis was satisfactory,29 much effort was devoted to improvingthe sensitivity of the assay. An amplification system using labeledsecondary antibodies was a promising technique for improvingthe sensitivity of immunoassay.30-31 The reactive activity of asecondary antibody cannot be significantly influenced by thebinding of small probe molecule. Meanwhile, the secondaryantibody can be multivalently bound to a primary antibody to forma constant immunological complex.

In this research, fluorescein-labeled secondary antibody wasutilized with CE-LIF for the determination of E2. A thermallyreversible hydrogel pNIPA was used as a dynamic modifier inthe capillary electrophoresis. A capillary electrophoretic immuno-assay of free E2 was then established. This immunoassay wasbased on the competitive reaction of antigen and free E2 with alimited amount of monoclonal antibody, with FITC-labeled second-ary antibody as a tracer. Both capillary zone electrophoresis andhydrogel-modified capillary electrophoresis were valued for suit-ability for free E2 determination. The employment of hydrogelenhanced the separation, and the fluoresceinated secondaryantibody effectively improved the sensitivity of analysis. Thismethod has a good potential in the determination of E2 inbiological fluids and environmental samples.

MATERIALS AND METHODSApparatus. The experiments were performed using a capillary

electrophoresis system of the P/ACE 5000 series equipped witha laser-induced fluorescence (LIF) detector obtained from Beck-man-Coulter Instruments (Fullerton, CA). A 488-nm argon ionlaser provided fluorescence excitation. The fluorescence intensitywas measured after passing through a 488-nm cutoff and a 520-nm 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 thedetection window, was employed for the separation. E2 and bovineserum album (BSA) were obtained from Sigma (St. Louis, MO).2-Propanol, acrylonitrile, ammonium persulfate (APS), and N,N,N′N′-tetramethylethylenediamine (TEMED) were purchased fromBeijing Chemicals (Beijing, China). Fluorescein isothiocyanate was

supplied by Zhaohui Pharmaceutics (Beijing, China). Fluoresceinisothiocyanate-labeled secondary antibody (Ab2*) was purchasedfrom Huamei Biochemicals (Beijing, China). N-isopropylacryl-amide was synthesized in our laboratory. Running buffer contain-ing 100 mmol/L Tris borate (pH 8.0), 0.5 mol/L sodium carbonatesolution (pH 9.5), and other solutions were prepared by dissolvingthe reagents in pure water supplied by a Milli-Q Plus waterpurification system (Millipore, Bedford, MA). The antigen (E2-BSA) was synthesized by coupling E2 with BSA (Mr, 68 000), andthe monoclonal antibody (Ab) was prepared and purified fromascites collected from immunized mice (Monoclonal Laboratories,Department of Biology, Peking University, China).

Preparation of Calibration Curve. Into nine identical samplescontaining 49.4 ng/mL antigen, 0.6 µg/mL monoclonal antibody,and 2.1 µg/mL labeled secondary antibody were added differentconcentrations of estradiol. Final concentrations of estradiol were0, 20, 25, 30, 50, 200, 600, 1500, and 5000 pg/mL. Each samplewas determined 5 times at the same concentration.

Procedures of Capillary Electrophoretic Immunoassay(CEIA). The poly-N-isopropylacylamide (pNIPA) was synthesizedaccording to our previous work.30 A fixed amount of pNIPA wasadded to Tris borate buffer of a suitable pH and concentration,which was subsequently packed into an uncoated capillary. Themixture of E2, E2-BSA, Ab, and Ab2* was injected into the positiveend with the pressure at 20 psi for 5 s, and then it was separatedin temperature control mode. The concentrations of Ab2*, E2-Ab-Ab2*, and Ag-Ab-Ab2* were calculated by peak areas, andthe concentration of E2 could be calculated.

Immunoassay Principle of Secondary Antibody System.Competitive and noncompetitive models are basic analyticalpatterns in the immunoassay. In the competitive model, at least akind of analytical reagent is limited. In our experiment, estradiolwas determined by competitive immunoassay with labeled second-ary antibody. E2-BSA (Ag) and estradiol (E2) were added to thelimited monoclonal antibody (Ab) solution and incubated for 30min at 37 °C. Then labeled secondary antibody(Ab2*) was addedto the above solution and incubated for 60 min at 37 °C. The wholeprocedure is illustrated in the following equations:

RESULTS AND DISCUSSIONOn the assumption that some parameters, such as column

length and temperature were held constant, parameters affectingresolution and speed of separation, including (i) ionic strength ofthe buffer, (ii) concentration of hydrogel in the buffer, and (iii)applied voltage were studied. Separation conditions were optimizedfor the study in this immunoassay.

Effect of Incubation Time. The type of separation was ahydrogel capillary electrophoresis in which labeled secondaryantibody (Ab2*) and monoclonal antibody(Ab) were added to asample. In this experiment, five identical samples containing 0.6µg/mL of antibody and 2.1 µg/mL of labeled secondary antibodywere incubated for different lengths of time. The incubation timehad a great influence on the binding rate of Ab2* to Ab. Times of10∼90 min were studied (Figure 1). When the incubation time is10 min, the binding rate is 15%. Increasing the incubation timeincreased the binding rate. When the incubation time wasprolonged to 60 min, the binding rate reached 65%. The binding

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

Ag-Ab + E2-Ab + Ag + E2 + Ab2* f

Ag-Ab-Ab2* + E2-Ab-Ab2* + Ab2* + Ag + E2 (2)

Analytical Chemistry, Vol. 73, No. 22, November 15, 2001 5617

rate was constant, even when the incubation time was over 60min. The results showed the best incubating time for Ab2* andAb was 60 min.

Affinity Constant in Solution of Ab-Ab2 Complexes.During analysis in the capillary electrophoresis immunoassay, thestability of the Ab-Ab2 complex must be maintained. This stabilityof the complex was characterized by the affinity constant insolution, which was measured by enzyme-linked immunosorbentassay (ELISA). In this experiment, monoclonal antibody (Ab) atvarious concentrations (3.71 × 10-10 to 1.90 × 10-7 mol/L) wasfirst incubated in 0.01 mol/L PBS (pH 7.4) with HRP-labeledsecondary antibody (Ab2*) at a constant concentration (0.4 µg/mL) at 37 °C. After the equilibrium was reached, 100 µL of eachmixture was transferred into the wells of microtitration platecoated with Ab (100 µL per well, at 8 µg/mL in 0.05 mol/L pH9.6 sodium carbonate, overnight at 4 °C), followed by an incubationfor 1 h at 37 °C. After the washing procedure and an addition ofenzyme substrate, the measured optical density determined thebound Ab2-HRP, which subsequently quantified the concentrationof free Ab2-HRP. Then the affinity constant was calculatedaccording to the Scatchard equation presented by Friguet et al.32

The affinity constant in solution was 3.43 × 107 L/mol, whichensured the Ab-Ab2* to be consistent in the capillary electro-phoresis immunoassay.

Effect of the pH and the Concentration of the Buffer. Theeffect of the different buffer ionic strengths on resolution wasstudied by CZE. For a given set of conditions, the concentrationof the buffer has important effects on resolution (Rs) and migrationtime (tm). When the buffer concentration increased, tm and Rs forEOF increased. The buffer containing 100 mmol/L Tris-boratewas selected for immunological reaction. It is important, for thesake of resolution, time of experiment, and Joule heating, to payattention to the ionic strength.

The pH of the buffer has an important effect on the surfacecharacteristics of the fused-silica capillary and the effective electriccharge of the ion. Because the hydrogel polymers swelled inaqueous solution and allowed permeation of different-molecular-weight compounds, an equilibrated state was attained betweenthe hydrogel polymer layers and the buffer solution, whicheliminated the interactions between proteins and the capillarysurface and allowed highly efficient and reproducible capillaryelectrophoretic separations of proteins. The pH of the buffer hasa large effect on the conformation of antigen and antibody.

Effect of Hydrogel Concentration. With many hydrophobicgroups in their structures, Ab2* and its complex would beadsorbed to the inner wall of the capillary, which would causefluctuation of the migration time. Hydrogel was added in our study.Figure 2 shows that resolution and migration time increased asthe gel concentration increased. The results (Figure 2) show thatresolution of Ab2* and the Ab2*-Ab compound was well-improved.The hydrogel we used was poly-N-isopropylacrylamide, whichcould form a linear hydrophobic internal space under certainconditions and decrease the interaction of Ab2*and its complexwith the inner wall. With the addition of hydrogel, an uncoatedfused-silica capillary can be applied very conveniently in theseparation of the immunoassay system.

Optimization of Immunological Reactions. Optimization ofimmunological reactions was performed. In this experiment, amixture with Ab2*, E2-Ab-Ab2*, Ag-Ab-Ab2*, Ag, and E2 wasinjected into a capillary. The optimized concentrations of antigen,monoclonal antibody, and labeled secondary antibody determinedin solution were 49.4 ng/mL, 0.6 µg/mL, and 2.1 µg/mL,respectively.

Immunological Reaction. In our experiments, capillaryelectrophoresis with laser-induced fluorescence detector (CE-LIF)using a replaceable hydrogel was demonstrated to be a usefultechnique for performing the separation step required in animmunoassay of estradiol. This type of immunoassay is a competi-tive assay in which antigen (E2-BSA), monoclonal antibody, andlabeled secondary antibody are added to a sample. Analyte, inthis case, unlabeled hapten (estradiol), of different concentrationscompetes with the antibody to form immunological complexes.The ratios of bound and free FITC-Ab2 changed according todifferent estradiol concentrations in the samples (Figure 3), andthe changes in area ratios of two peaks were consistent withestradiol concentrations. The calibration curve (Figure 4) forestradiol can be acquired by plotting the relative area over themaximum area ratio (the area ratio of bound to free tracer at zeroconcentration of estradiol) against the log concentrations of

(32) Friguet, B.; Chaffotte, A. F.; Djavadi-Ohaniance, L.; Goldberg, M. E. J.Immunol. Methods 1985, 77, 305-319.

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

Figure 2. Separation of bound secondary antibody and freesecondary 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.

5618 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

estradiol. The linear range (n ) 8, r ) 0.995) and detection limitfor estradiol were ∼25 pg/mL to 5ng/mL and 9.0 pg/mL,respectively.

Because the analogues of estradiol would interfere with thedetermination of estradiol in some methods, it is necessary toestimate the interferences of them in the study. The possibilityof interference with the immunological process from analoguescan be estimated from the cross-reaction. The cross-reactions ofanalogues of estradiol, determined by ELISA, are estrone, 2.4%;estriol, 4.7%; and progesterone, 1.3%. The results show that therewas no interference from those analogues in the determinationof estradiol at the applied concentration level.

Analytical Application. Simulated samples were analyzed bythis method. Two samples, one with low concentration (150 pg/mL) and the other with high concentration (1.0 ng/mL) ofestradiol, were diluted by a de-hormoned serum from healthyvolunteers. Seven repetitive experiments were made under thesame conditions. The analytical results are listed in Table 1.Results show that this method could be applied satisfactorily as areliable means for detecting estradiol in serum.

CONCLUSIONSWith the thermal reversible hydrogel and labeled secondary

antibody, a simple and rapid capillary electrophoretic immuno-assay of estradiol was demonstrated in an uncoated fused-silica

capillary. Adding hydrogel into the buffer can minimize theadsorption of antibody, antigen, secondary antibody, and theircomplexes to the inner wall of capillary.

These results have demonstrated that secondary antibody canbe applied to separate estradiol rapidly and efficiently in thecapillary packed with replaceable poly-N-isopropylacrylamide(pNIPA) hydrogel. This method can be employed to independentlydetect estradiol in serum at any time. Even though this technique’sdetection 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 someclinic analyses when the concentration of estradiol is abnormallyhigh.

ACKNOWLEDGMENTThe authors acknowledge financial support from the National

Natural Science Foundation of China (20075003, 20075001) andExcellent Young Teacher Program from MOE, P.R.C. We alsothank Dr. Peter Osborne for his helpful discussion.

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

AC010537A(33) Worthman, C. M.; Stallings, J. F.; Hofman, L. F. Clin. Chem. 1990, 36,

1769-1773.

Figure 3. CE-LIF immunoassay of estradiol using 1.25 mg/mLhydrogel: 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.

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

Table 1. Recovery of Samples Using 1.25 mg/mLHydrogel Capillary Electrophoresis

sample content(ng/mL)

added(ng/mL)

detection(ng/mL, n ) 7)

RSD(n ) 7)

recovery(%, n ) 7)

0 0.15 0.138 2.57 92.00 1.0 0.923 2.16 92.3

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