Precolumn Affinity Capillary Electrophoresis for the Identification of

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Anal. Chem. 1998, 70, 5339-5343

Correspondence

Precolumn Affinity Capillary Electrophoresis for the Identification of Clinically Relevant Proteins in Human Serum: Application to Human Cardiac Troponin I Joseph J. Dalluge* and Lane C. Sander

Analytical Chemistry Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-0001

An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (∼5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femtomolar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed. The accurate measurement of clinically important disease-state marker proteins using conventional immunoassays is confounded by several factors, including assay matrix effects, differences in antisera binding specificities, and a lack of universally accepted standards. The current interest in the accurate determination of these analytes necessitates the development of highly sensitive and selective methods for their measurement which overcome the limitations described above and facilitate development of certified reference materials for these proteins independently of any manufacturer’s immunoassay. The use of capillary electrophoresis (CE) as an approach to the determination of clinically relevant proteins in biological fluids

is attractive due to its characteristically high efficiency, mass sensitivity, and resolution, as well as its compatibility with a wide range of detectors. The considerable complexity of biologically derived mixtures, however, makes the direct determination of trace components in these matrixes using CE impractical from the standpoint of selectivity. To overcome the problems of low concentration sensitivity and selectivity in the analysis of complex mixtures using CE, a number of investigators have reported the use of chromatographic sorbents within fused silica capillary cartridges1-8 for the selective preconcentration of specific analytes. These methods can be divided into techniques that utilize adsorptive phases which preconcentrate with relatively low selectivity (e.g., C183,4), potentially obscuring analytes of interest present at low concentrations, and those that interact with high selectivity and affinity (e.g., supports that exhibit specific immunological properties) for a specific analyte within such a matrix.1,5 Reported methods in the latter category include those utilizing antibodies immobilized on the capillary wall, glass beads, or solid glass rods6-8 and those within a packed bed of protein G-coupled silica.5 Notable limitations of these approaches include low accessible surface area for analyte binding,6-8 failure of preconcentration cartridges due to blockage,6 the need for off-line application of samples,7 the simultaneous elution of antibody and antigen,5 and the need to physically couple via a polyethylene sleeve the preconcentration cartridge to a separation capillary.5-8

* To whom correspondence should be addressed. Fax: 301-977-0685. Email: [email protected].

(1) Guzman, N. A.; Park, S. S.; Schaufelberger, D.; Hernandez, L.; Paez, X.; Rada, P.; Tomlinson, A. J.; Naylor, S. J. Chromatogr. B 1997, 697, 37-66. (2) Tomlinson, A. J.; Guzman, N. A.; Naylor, S. J. Cap. Electrophor. 1995, 2, 247-266. (3) Strausbauch, M. A.; Landers, J. P.; Wettstein, P. J. Anal. Chem. 1996, 68, 306-314. (4) Hoyt, A. M.; Beale, S. C.; Larmann, J. P.; Jorgenson, J. W. J. Microcolumn Sep. 1993, 5, 325-330. (5) Cole, L. J.; Kennedy, R. T. Electrophoresis 1995, 16, 549-556. (6) Guzman, N. A.; Trebilcock, M. A.; Advis, J. P. J. Liq. Chromatogr. 1991, 14, 997-1015. (7) Guzman, N. A. J. Liquid Chromatogr. 1995, 18, 3751-3768. (8) Tomlinson, A. J.; Benson, L. M.; Guzman, N. A.; Naylor, S. J. Chromatogr. A 1996, 744, 3-15.

10.1021/ac980773u Not subject to U.S. Copyright. Publ. 1998 Am. Chem. Soc.

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Published on Web 11/04/1998

This paper reports a novel approach to the selective determination of a clinically relevant protein in human serum which overcomes the limitations described above. We have designated this approach “precolumn affinity capillary electrophoresis (PACE)”. The method represents the first reported utilization of a short packed bed of porous silica containing covalently immobilized monoclonal antibodies directly integrated within a single separation capillary for the isolation, resolution, and identification of specific proteins in complex biologically derived mixtures. The utility of this method is demonstrated for the trace enrichment, separation, and identification of human cardiac troponin I (cTnI) in human serum. Cardiac troponin I is a 24kDa structural protein that interacts with troponin C and troponin T as part of a complex essential for contraction of striated muscle in cardiac tissue.9 It is rapidly released into the bloodstream following myocardial infarction and has become the preferred diagnostic marker for such an event.10 This report represents an investigation into the potential of PA-CE for the selective determination of cTnI toward standardization of the clinical measurement of this protein in human serum. EXPERIMENTAL SECTION Note: Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the material or equipment identified are the best available for the purpose. Chemicals. Urea, Tween-20, sodium phosphate, Tris, sodium chloride, potassium chloride, ethylenediaminetetraacetic acid (EDTA), β-alanine, and β-mercaptoethanol were purchased from Sigma Chemical Co. (St. Louis, MO). Acetic acid was purchased from J.T. Baker (Phillipsburg, NJ). Glutaric dialdehyde and 3-aminopropyltriethoxysilane were purchased from Aldrich Chemical Co. (Milwaukee, WI). Monoclonal anti-cardiac troponin I, clone 7F4, was purchased from Research Diagnostics Inc. (Flanders, NJ). Cardiac troponin I was purchased from Calbiochem (La Jolla, CA) and Advanced Immunochemical Inc. (Long Beach, CA). Human serum was obtained from Interstate Blood Bank Inc. (Memphis, TN). Buffer Preparation. The separation buffer utilized for all CE experiments was 25 mmol/L β-alanine, 125 mmol/L acetic acid, pH 3.8. The binding buffer was 200 mmol/L phosphate, pH 7.4. The elution buffer consisted of 35 mmol/L Tris-acetate, 7 mmol/L β-mercaptoethanol, 5 mmol/L EDTA, 2 M urea, pH 3.5. All solutions were routinely filtered using a 0.2-µm nylon filter and degassed by sonication prior to use. Covalent Immobilization of Anti-Cardiac Troponin I Antibodies on Porous Silica. To a precisely known amount of Nucleosil porous silica in an eppendorf tube (10 µm diameter, 4000-Å pores), was added 200 µL of a 10% solution of 3-aminopropyltriethoxysilane in acetone. This particular silica was chosen because larger silica particles containing 4000 Å pores which will allow perfusive flow are not commercially available. The mixture was incubated at 45 °C for 12 h with periodic mixing. The silica (9) Mair, J.; Dienstl, F.; Puschendorf, B. Crit. Rev. Clin. Lab. Sci. 1992, 29, 31-57. (10) Cummins, B.; Auckland, M. L.; Cummins, P. Am. Heart J. 1987, 113, 13331344.

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Figure 1. Apparatus used for packing precolumn affinity capillary electrophoresis columns.

was then washed three times with 300 µL of acetone and allowed to air-dry. Glutaric dialdehyde (200 µL of a 5% aqueous solution) was added to the dry silica and incubated at room temperature for 4 h. The silica was washed three times with 300 µL of 200 mmol/L phosphate buffer, pH 7.4. Monoclonal anti-cTnI (∼1 mg) in the same buffer was added to the silica and allowed to stand for 1 h at room temperature with occasional mixing. The silica containing the covalently immobilized antibody (anti-cTnI silica) was finally washed three times with 200 µL of 20 mmol/L TrisCl, 137 mmol/L NaCl, 3 mmol/L KCl, containing 0.05% (v/v) Tween-20 nonionic detergent, pH 7.2, to remove any nonspecifically bound protein. The amount of monoclonal anti-cardiac troponin I coupled to the silica was estimated on the basis of the protein concentration in the combined supernatants of the washes using the Bio-Rad DC protein assay (Hercules, CA). The amount of antibody coupled to the silica was 0.45 µmol/g. The capture efficiency of the prepared anti-cTnI silica for cTnI was evaluated by reacting cTnI with the silica in amounts proportional to those which would be encountered in subsequent on-line PA-CE experiments and measuring the amount of unbound protein in the resulting supernatant. The capture efficiency calculated in this manner was >90%. Recovery of the cTnI from the silica with elution buffer was also calculated and found to be >90%. Preparation of Precolumn Affinity Capillary Electrophoresis (PA-CE) Column. Precolumn affinity capillary columns were prepared using procedures modified from those used for capillary liquid chromatography11 and capillary electrochromatography (CEC).12,13 The apparatus used is shown in Figure 1. CElect-N (75 µm i.d. × 360 µm o.d.) coated fused silica capillary tubing was obtained from Supelco Inc. (Bellefonte, PA). For preparation of a 50-cm column, a length of capillary of approximately 60 cm was employed. A semipreparative cartridge guard column (Upchurch Scientific, Oak Harbor, WA) was modified for use as a slurry reservoir. One internal frit was removed to permit passage of silica into the capillary tubing. A stir bar was placed in the reservoir to suspend the silica during column packing. Ap(11) Dalluge, J. J.; Nelson, B. C.; Thomas, J. B.; Welch, M. J.; Sander, L. C. Rapid Commun. Mass Spectrom. 1997, 11, 1753-1756. (12) Boughtflower, R. J.; Underwood, T.; Patterson, C. J. Chromatographia 1995, 40, 329-335. (13) Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-657.

proximately 5 mg of anti-cTnI silica was slurried in 2 mL of 20 mmol/L Tris-Cl, 137 mmol/L NaCl, 3 mmol/L KCl, containing 0.05% (v/v) Tween-20, at pH 7.2. It is notable that the presence of the nonionic detergent in the slurry mixture prevented aggregation of the packing material and plugging of the column during the subsequent pressurization. The slurry was placed in the reservoir and the system pressurized to 40-60 MPa with the same buffer using a LC chromatographic pump. After the fused silica tubing was filled (10 min), the pressure was reduced to 13 MPa, and internal frits were placed 5 mm apart at the inlet end of the capillary by sintering the packing material with a fiber-optic fusion splicer (Power Technologies, Inc., Little Rock, AR) (Figure 1). Arc conditions were set at 14 mA for 1.5 s. Following formation of the frits, the column was reversed and repressurized to remove from the capillary any remaining silica not contained within the precolumn. The column was finally depressurized, cut at the inlet frit using a ceramic wafer column tool, and installed into the CE instrument. The PA-CE columns developed for use in our laboratory provided long-term stability (several weeks at room temperature) to the antibodies for repeated use in the determination of cTnI. Capillary Electrophoresis Instrumentation and Conditions. Capillary electrophoresis experiments were performed on a Thermo Capillary Electrophoresis Crystal CE system, model 310 (Franklin, MA), directly coupled to a variable-wavelength UV absorbance detector. Absorbance was monitored at 280 nm for all experiments. Samples were analyzed in separation buffer using a 75-µm- i.d. × 360-µm- o.d. × 67-cm CElect-N coated capillary (off-line immunoaffinity capture experiments) or the PA-CE column described above. During the electrophoretic runs, a constant 16 kV was applied across the capillary. Determination of cTnI in Human Serum Using Off-Line Immunoaffinity Capture. The extraction of cTnI from human serum utilizing the anti-cTnI silica was demonstrated by incubating a cTnI-spiked serum sample (100 nmol/L cTnI) with a small amount of anti-cTnI silica conditioned in binding buffer. The supernatant was decanted, and the silica was washed three times with CE running buffer. Cardiac troponin I was then eluted from the silica with a small amount of elution buffer and run using the CE conditions described above. The identity of cTnI extracted from serum was verified by comparison of its migration time with that of a cTnI standard, as well as by matrix-assisted laser desorption/ionization mass spectrometry. Procedure for the Determination of Cardiac Troponin I in Human Serum Using On-Line PA-CE. To demonstrate the efficacy of measuring cardiac troponin I in biological fluids using PA-CE, a known amount of cTnI was spiked in 1 mL of human serum to final concentrations ranging from 2 to 80 nmol/L. The column was conditioned for binding cTnI in serum by rinsing with three column volumes of binding buffer prior to sample injection. After sample injection by pressure at the inlet (approximately 1020 ng of cTnI injected), the PA-CE capillary was rinsed extensively with running buffer to eliminate any nonspecifically bound serum proteins, salts, and other impurities from the precolumn. Cardiac troponin I was then selectively eluted from the precolumn with a short plug of elution buffer and run directly using the CE conditions described above.

Figure 2. Identification of cTnI in human serum using off-line immunoaffinity capture. (A) Electropherogram of cTnI extracted from a spiked human serum sample using anti-cTnI silica. (B) Electropherogram of a cTnI standard dissolved in elution buffer. Separation buffer, 25 mmol/L β-alanine, 125 mmol/L acetic acid, pH 3.8; capillary, 75 µm × 67 cm.

RESULTS AND DISCUSSION The specific aim of this study was the hybridization of affinity chromatography, capillary electrochromatography microfabrication technology, and free solution capillary electrophoresis to improve on existing CE methods for the enhancement of selectivity and sensitivity in the determination of proteins from complex matrixes. As such, the term precolumn affinity-capillary electrophoresis has been used to reflect the hybrid nature of this technique while distinguishing it from existing preconcentration CE techniques. To address the determination of low levels of the disease-state marker protein cardiac troponin I in human serum, we have investigated the use of a packed-bed of perfusive stationary phase containing covalently immobilized anti-cTnI antibodies as a precolumn within the separation capillary as a means for maximizing selectivity and concentration sensitivity for the identification of this protein. The results of this investigation demonstrate the potential of PA-CE for the separation and identification of clinically important proteins in a serum matrix at physiologically relevant levels. Selective Extraction of cTnI from Serum Using Anti-cTnI Silica. Prior to examining PA-CE for the identification of cTnI in complex matrixes, the efficacy of extracting this protein from human serum off-line using the prepared anti-cTnI silica was tested. The results of this experiment are illustrated in Figure 2. A human serum sample spiked with cTnI was applied to the anticTnI silica in binding buffer. After extensive rinsing with the CE running buffer, the cTnI was subsequently desorbed from the antibody and run on a neutrally coated capillary column. A typical electropherogram of cardiac troponin I extracted from human serum in this manner is illustrated in Figure 2A. For comparison, an electropherogram of a cTnI standard dissolved in elution buffer is shown in Figure 2B. The identity of troponin I was further confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of a collected sample corresponding to the peak in Figure 2A (data not shown). Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

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Figure 4. Determination of cTnI at a clinically relevant concentration in human serum. The sample (2 nmol/L) was injected hydrodynamically, washed, eluted, and run directly as described in the Experimental Section. CE conditions were the same as those described for Figure 3.

Figure 3. Identification of cTnI in human serum using on-line precolumn affinity capillary electrophoresis. (A) Electropherogram of cTnI selectively extracted from a spiked human serum sample using PA-CE. (B) Electropherograms of sample blanks: (1) water (no cTnI); (2) human serum (no cTnI). (C) Electropherogram of a cTnI standard applied to a PA-CE column. Samples were injected hydrodynamically, washed, eluted, and run directly as described in the Experimental Section. Separation buffer, 25 mmol/L β-alanine, 125 mmol/L acetic acid, pH 3.8; capillary, 75 µm × 50 cm, containing the 5-mm anticTnI affinity precolumn.

The results of this experiment demonstrate that cTnI can be applied as part of a complex biologically derived matrix to anticTnI silica, selectively retained while the solid phase is washed with aqueous electrophoresis buffer, and successfully eluted for subsequent CE determination. Separation and Identification of cTnI in Human Serum by PA-CE. While off-line extraction of a clinically relevant protein from serum using antibody-coupled silica may be a useful approach under certain circumstances, it has several potential limitations, including analyte losses to surfaces and protein denaturation caused by sample manipulation. These destructive effects are especially undesirable when working with samples in which the analyte of interest is present at low concentrations. For this reason, the on-line determination of cTnI in serum utilizing a packed capillary precolumn is attractive due to reduced sample handling and subsequently improved method sensitivity. A demonstration of the potential utility of PA-CE for the separation and identification of cTnI in human serum is illustrated in Figure 3. First, a serum sample spiked with troponin I (80 nmol/L) was loaded onto the precolumn affinity capillary under high pressure (2000 mbar, approximately 20 ng cTnI), rinsed with several column volumes of running buffer, eluted from the anticTnI packing material by positive pressure with elution buffer, and run directly by CE (320 V/cm, 11 µA). The volume of elution buffer used for the desorption of cTnI into the separation region 5342 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998

of the PA-CE capillary was found to be important with regard to the efficiency of the CE separation. The amount of elution buffer injected was determined empirically for each column to minimize band broadening. These amounts varied between different columns due to differences in flow rates through the precolumns. The resulting electropherogram is shown in Figure 3A. After the appropriate sample blanks were run (Figure 3B) to demonstrate the absence of carry-over in the PA-CE system during sequential analyses, a cTnI standard was run under identical conditions (Figure 3C) to verify the identity of the peak in Figure 3A. The application of PA-CE to the identification of cTnI in serum at clinically relevant levels (∼2 nmol/L)14 was also accomplished. This determination is illustrated in Figure 4. Further, the measurement of cTnI derived from its complexed form (cTnTI-C) in serum was performed by initial disruption of the complex in 10 mmol/L EDTA followed by PA-CE analysis. The resulting electropherogram (not shown) was identical to that illustrated in Figure 4. Precision and linearity for multiple determinations of cTnI with varying amounts of sample injected was within the range 3-4%. It is clear from Figures 3 and 4 that the on-line sample cleanup, selectivity, sensitivity (limit of detection < 10 ng, concentration limit of detection < 2 nmol/L), efficiency, and sample recovery of this method are remarkable. The only limitation of PA-CE with regard to the determination of proteins at trace levels is the low flow rate through the capillary (1-5 µL/ min) when using hydrodynamic injection with a commercial CE instrument. This requires a significant period of time for sample application at clinical levels prior to the analysis (∼60 min for the cTnI determination illustrated in Figure 4). We are currently investigating ways to overcome this limitation through use of larger diameter porous silica particles (20-30 µm) and incorporation of a flow-gated LC-to-CE interface5,15 for more rapid sample application. Advantages of PA-CE. The PA-CE system described herein has the following notable advantages over previously described immunoaffinity CE techniques: (1) The use of a packed bed of antibody-containing silica, in addition to the large channel diameter of the solid support which allows analyte transport into pore spaces, increases the effective accessible surface area for analyte binding compared to precolumn cartridges containing antibodies immobilized on the capillary wall, glass beads, or solid glass rods.6-8 (2) The use of a uniformly packed and perfusive solid (14) Wu, A. H.; Feng, Y. J.; Moore, R.; Apple, F. S.; McPherson, P. H.; Buechler, K. F.; Bodor, G. Clin. Chem. 1998, 44, 1198-1208. (15) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 576-581.

support16 prevents failure of the PA-CE column due to blockage of the precolumn, known to occur with glass beads due to packing of the beads at the end frit. (3) The use of covalently immobilized antibodies allows the selective desorption of antigen from the precolumn, resulting in an electropherogram with a single, easily identified peak (Figures 3 and 4). By comparison, a previously described immunoaffinity CE method,5 which utilizes a packed bed of protein G-coupled antibodies, is confounded by simultaneous elution of antibody and antigen, making interpretation of the subsequent electropherogram difficult. (4) The use of an affinity precolumn directly integrated within the separation capillary makes the currently described technology compatible with a wide variety of commercial CE instrumentation. CONCLUSIONS The technology described in this report represents a mixedmode technique which is an outgrowth of microfabrication efforts in capillary electrochromatography and selectivity enhancement efforts in capillary electrophoresis. It should be noted that the utility of capillaries fabricated with integral packed beds is not limited to the determination of proteins in complex matrixes but could be expanded to include such applications as precolumn microreactor beds for on-line digestion of nucleic acids and proteins, nonspecific solid-phase extraction, or multidimensional separations utilizing microscale liquid chromatography and capillary electrochromatography. (16) Li, D.; Remcho, V. T. J. Microcolumn Sep. 1997, 9, 389-397.

Finally, with concentration values of disease-state marker proteins such as cardiac troponin I differing by as much as an order of magnitude between different immunoassay kits,16 the need for standardization of diagnostic immunoassays has become a priority within the clinical community. This report describes a reliable system for the efficient determination of cTnI in human serum using precolumn affinity capillary electrophoresis. This technique, together with the use of an appropriate internal standard, will be the basis of a method for the accurate quantification of cTnI in human plasma. Such a method will facilitate the development of a certified reference material for this protein which will aid in the validation and calibration of existing clinical immunoassays. Further, the approach described herein should be applicable to the simultaneous measurement of multiple antigens, as well as the measurement of other disease-state marker proteins, including prostate-specific antigen (PSA) and R-fetoprotein (AFP), for which the variability of results among current clinical methods is high. ACKNOWLEDGMENT J.J.D. acknowledges support by the NIST/NRC Postdoctoral Research Associateships Program.

Received for review July 14, 1998. Accepted September 29, 1998. AC980773U

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