Noncompetitive Immunoassay of Small Analytes at the Femtomolar

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Anal. Chem. 2000, 72, 5779-5786

Noncompetitive Immunoassay of Small Analytes at the Femtomolar Level by Affinity Probe Capillary Electrophoresis: Direct Analysis of Digoxin Using a Uniform-Labeled scFv Immunoreagent Frank T. Hafner, Roger A. Kautz, Brent L. Iverson,† Roger C. Tim,‡ and Barry L. Karger*

Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Sitedirected mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-µm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 °C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A “mix-andinject” assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes. Solid-phase immunoassays, introduced in 1966,1 have become broadly used for trace analysis.2 Separation of bound from unbound label by washing is a simple method for heterogeneous immunoassay, and the ability to perform multiple assays in parallel on microtiter plates has offered high throughput. In the noncompetitive format (see next paragraph), the two-site immunometric assay (sandwich assay) has shown limits of detection below 0.1 pM (10-13 M) for proteins and other high molecular weight analytes that are large enough to bind two antibodies simultaneously.3,4 † Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712. ‡ Current address: Biocept Inc., Carlsbad, CA, 92009. (1) Wide, L.; Porath, J. Biochim. Biophys. Acta 1966, 130, 257-260. (2) Christopoulos, T. K.; Diamandis, E. P. In Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Academic Press: San Diego, 1996; p 227.

10.1021/ac000853+ CCC: $19.00 Published on Web 10/26/2000

© 2000 American Chemical Society

[Several nomenclatures introduced earlier have become ambiguous with further innovation. The significant distinction at this time is between competitive and noncompetitive assay formats, as defined by Ekins.5 Competitive assays measure the number of antibody sites remaining unbound after a defined and limiting amount of antibody is incubated with an unknown sample. The signal is inversely proportional to analyte concentration in a sigmoidal calibration curve, with a narrow linear range near its midpoint. A noncompetitive assay measures the number of analytebound antibody sites, producing a signal linearly proportional to analyte concentration over several orders of magnitude. A direct assay uses labeled antibody. Direct assays include most noncompetitive assays and also a “direct competitive” format which measures the amount of labeled antibody not blocked from binding to immobilized analyte after incubation with an unknown sample. In a heterogeneous assay, excess tracer (labeled antibody or labeled analyte) is separated from tracer bound in the immune complex to be quantitated. Heterogeneous assays may be competitive or noncompetitive. A homogeneous assay does not require such separation, for example, fluorescence polarization.6 With one exception,7 homogeneous assays are exclusively competitive.] In contrast to the sensitivity of sandwich assays, detection of low molecular weight analytes, including most drugs and hormones, must rely on competitive assays.5 Noncompetitive assays are typically 100-fold more sensitive than competitive assays8 and provide a linear range up to 5 orders of magnitude.9 Efforts have been made to extend the sandwich format to small analytes using cross-linking,8 blocking-and-replacement,10,11 or anti-idiotype antibodies.12,13 However, these methods have limited application or (3) Cook, D. B.; Self, C. H. Clin. Chem. 1993, 39, 965. (4) O’Connor, T.; Gosling, J. P. J. Immunol. Methods 1997, 208, 181-189. (5) Ekins, R. P. J. Chem. Educ. 1999, 76, 769-780. (6) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (7) Ullman, E. F. J. Chem. Educ. 1999, 76, 781-788. (8) Pradelles, P.; Grassi, J.; Creminon, C.; Boutten, B.; Mamas, S. Anal. Chem. 1994, 66, 16-22. (9) Taran, F.; Bernard, H.; Valleix, A.; Creminon, C.; Grassi, J.; Olichon, D.; Deverre, J. R.; Pradelles, P. Clin. Chim. Acta 1997, 264, 177-192. (10) Giraudi, G.; Anfossi, L.; Rosso, I.; Gabbiani, C.; Giovannoli, C.; Tozzi, C. Anal. Chem. 1999, 71, 4697-4700. (11) Freytag, J. W.; Dickinson, J. C.; Tseng, S. Y. Clin. Chem. 1984, 30, 417420. (12) Self, C. H.; Dessi, J. L.; Winger, L. A. Clin. Chem. 1994, 40, 2035-2041. (13) Mares, A.; De Boever, J.; Osher, J.; Quiroga, S.; Barnard, G.; Kohen, F. J. Immunol. Methods 1995, 181, 83-90.

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performance, and all require additional steps. A simple noncompetitive immunoassay format applicable to small analytes remains an unmet need in trace analysis.4,10 Capillary electrophoresis (CE) has recently emerged as an alternative to immunosorbent methods for heterogeneous immunoassays (IA).14 CE-IA are simple “mix-and-inject” protocols using nanoliters of reagents, have shown separation times of under 1 min;15,16 and can be integrated in lab-on-a chip systems.17 CE with laser-induced fluorescence (LIF) detection can detect 10-12 M labeled protein.18 Where immunosorbent assays are limited by the background of the tracer (labeled analyte or labeled antibody) binding nonspecifically to the well,5 in CE-IA the immune complex is separated from excess tracer in solution with the high resolution of CE. Affinity probe capillary electrophoresis (APCE) is a noncompetitive assay, using a single antibody (or other receptor).19-21 APCE has shown limits of detection of 1 pM human growth hormone, with capillary isoelectric focusing (cIEF) preconcentration.19 Recently, a drug, dorzolamide, was detected at 16 pM using labeled human carbonic anhydrase II (hCAII) as an affinity probe in a capillary zone electrophoresis (CZE) assay.22 APCE is in principle a general method. However, the need for electrophoretically homogeneous affinity probes, i.e., labeled receptors that migrate as a single sharp peak in CE analysis, can be problematic. Antibodies are particularly desirable as affinity probes because they can be generated against virtually any analyte. But native antibodies, and their active proteolytic fragments (Fab), are generally unsuitable for direct CE-IA because they are electrophoretically heterogeneous. A uniform, monovalent affinity probe was successfully prepared by reduction and partial oxidation of an antibody (Fab′)2 fragment,19 but this work required multiple purification steps, and the method was specific to the amino acid sequence of the starting material. The heterogeneity of antibodies is attributed to variability in glycosylation and to degradation such as deamidation both within the cell and after secretion.23 Single-chain antibody variable-region fragments (scFv), the smallest construction of an antibody that retains the complete binding site, are made by recombinant protein expression of the variable-region domains of the antibody heavy and light chains, VH and VL.24 Recombinant antibodies can be derived from existing monoclonal antibodies or can be generated de novo using phage display methods,25 importantly without the need for animal facilities. The addition of unique cysteine residues to scFv’s by (14) Schmalzing, D.; Nashabeh, W. Electrophoresis 1997, 18, 2184-93. (15) Tao, L.; Aspinwall, C. A.; Kennedy, R. T. Electrophoresis 1998, 19, 403408. (16) Koutny, L. B.; Schmalzing, D.; Taylor, T. A.; Fuchs, M. Anal. Chem. 1996, 68, 18-22. (17) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (18) Pinto, D. M.; Arriaga, E. A.; Craig, D.; Angelova, J.; Sharma, N.; Ahmadzadeh, H.; Dovichi, N. J.; Boulet, C. A. Anal. Chem. 1997, 69, 3015-3021. (19) Shimura, K.; Karger, B. L. Anal. Chem. 1994, 66, 9-15. (20) Shimura, K.; Kasai, K. Anal. Biochem. 1997, 251, 1-16. (21) German, I.; Buchanan, D. D.; Kennedy, R. T. Anal. Chem. 1998, 70, 45404545. (22) Tim, R. C.; Kautz, R. A.; Karger, B. L. Electrophoresis 2000, 21, 220-226. (23) Mimura, Y.; Nakamura, K.; Tanaka, T.; Fujimoto, M. Electrophoresis 1998, 19, 767-775. (24) Neri, D.; Petrul, H.; Roncucci, G. Cell. Biophys. 1995, 27, 47-61. (25) Vaughan, T. J.; Williams, A. J.; Pritchard, K.; Osbourn, J. K.; Pope, A. R.; Earnshaw, J. C.; McCafferty, J.; Hodits, R. A.; Wilton, J.; Johnson, K. S. Nat. Biotechnol. 1996, 14, 309-314.

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site-directed mutagenesis to provide specific attachment sites is routine.26 When scFv’s are expressed intracellularly in bacteria, they form insoluble aggregates called inclusion bodies, which can be readily purified, and functional protein can be recovered by controlled renaturation.27 For generation of uniform reagents, incorporation of nascent scFv into the aggregate may offer protection from degradation, which causes heterogeneity. The goal of this work was the development of a general approach for noncompetitive CE-IA based on APCE using uniform scFv reagents generated by recombinant antibody technology. As a model system, an scFv is used that binds digoxin with an affinity comparable to what is obtainable by phage display methods.25 Digoxin is a common model analyte,11,12,31 which facilitates comparison with other immunoassay formats. Digoxin is also small (781 Da) and uncharged, which is the most challenging class of analytes for noncompetitive CE-IA. The attractive features of this approach include high reproducibility and sensitivity, low sample consumption, and the possibility of even further miniaturization. APCE is shown to have the potential to be a general method for trace analysis of clinical and pharmaceutical samples of small molecules. MATERIALS AND METHODS Instrumentation. A P/ACE model 2210 equipped with a Laser Module 488 (Beckman-Coulter Instruments, Fullerton, CA) was used for all CE-LIF analyses. The excitation wavelength in LIF was 488 nm and the emission wavelength 520 nm. All separations were performed in polyimide-coated fused-silica capillaries (50 µm i.d., 360 µm o.d.; Polymicro Technologies, Phoenix, AZ), modified in-house with a poly(vinyl alcohol) (PVA) coating.32 Chemicals. Unless otherwise specified, all chemicals were reagent grade from Fisher Scientific (Fair Lawn, NJ). Fresh frozen serum, bovine serum albumin (BSA), nitrilotriacetic acid beaded agarose (IMAC resin), sinapinic acid, 3-(cyclohexylamino)-2hydroxy-1-propanesulfonic acid (CAPSO), digoxin, and calibrated digoxin standard solutions in serum were purchased from Sigma (St. Louis, MO). Additional digoxin standards were obtained from Behring Diagnostics (Cupertino, CA). 5-Iodoacetamidofluorescein (5-IAF) was from Molecular Probes (Eugene, OR). Digoxigeninconjugated DNA 30-mer (digDNA) was from Boehringer Mannheim (Indianapolis, IN). Oasis HLB solid-phase extraction cartridges were purchased from Waters (Milford, MA). All solutions were prepared using doubly deionized water (18 MΩ) purified by an Alpha-Q System (Millipore, Marlborough, MA). HPLC-grade ethanol and acetonitrile were from Fisher Scientific. Mutagenesis, Purification, and Labeling of Single-Chain Antibody. The parent expression plasmid used as starting material, consisting of the 26-10 scFv(dig) gene in the pET25 (26) Kipriyanov, S. M.; Dubel, S.; Breitling, F.; Kontermann, R. E.; Heymann, S.; Little, M. Cell. Biophys. 1995, 26, 187-204. (27) Burks, E. A.; Iverson, B. L. Biotechnol. Prog. 1995, 11, 112-114. (28) Lilie, H.; Schwarz, E.; Rudolph, R. Curr. Opin. Biotechnol. 1998, 9, 497501. (29) Nieba, L.; Honegger, A.; Krebber, C.; Pluckthun, A. Protein Eng. 1997, 10, 435-444. (30) Wall, J. G.; Pluckthun, A. Protein Eng. 1999, 12, 605-11. (31) Chen, F. T.; Pentoney, S. L., Jr. J. Chromatogr., A 1994, 680, 425-430. (32) Goetzinger, W.; Karger, B. U.S. Patent 5840388, Northeastern University, 1998.

bacterial expression vector, has been previously described.27,33 Mutagenesis, expression, and protein purification were made using standard procedures;34 specific details may be obtained from www.barnett.neu.edu/KargerRG/people/rKautz. N- or C-terminal cysteine codons were introduced into the scFv gene by PCR amplification using primers containing the desired additional nucleotides. The N-Cys and C-Cys variant genes were then subcloned back into pET25 (Novagen, Madison WI). A culture of bacteria transformed with each expression plasmid was stored at -80 °C in 10% glycerol (glycerol stocks). The mutant proteins were expressed in Escherichia coli, which were harvested by centrifugation and lysed by sonication. Inclusion bodies were purified by washing the lysed cell pellets with nonionic detergent to remove soluble components. Aliquots of the washed cells were pelleted and stored under 5 mM dithiothreitol at -80 °C. scFv was recovered from inclusion bodies by solubilizing the cell pellet in 8 M urea and mixing with Ni-charged immobilized metal affinity chromatography (IMAC) resin. The resin was washed with 8 M urea, and then the scFv was renatured on the resin by washing with a gradient from 8 M to 0 urea. The renatured scFv was reacted on the IMAC resin with the thiolreactive dye, 5-IAF (Molecular Probes). Excess dye was removed by exhaustive washing before the scFv was eluted from the resin in 50 mM EDTA in Fv buffer (90 vol % 50 mM TRIS-HCl pH 7.4, 50 mM KCl, 10% vol glycerol).27 The eluate was dialyzed against Fv storage buffer and then passed through a 0.02-µm filter (Anotop, Whatman, Clifton, NJ) which had been passivated with BSA. Protein concentration was determined by UV absorbance, 280 ) 53 000 M-1 cm-1. For each batch of labeled scFv, CE-LIF intensity was calibrated to protein concentration to permit measurement of scFv concentration in dilutions of the reagent. Labeled scFv stock solutions were stored in Fv storage buffer with 1 mg/ mL BSA. Sample Preparation. A 1 mg/mL stock solution of digoxin was made gravimetrically in HPLC-grade ethanol (Fisher Scientific) and confirmed by UV absorbance (220 ) 12 800, Merck Index; Merck and Co.: Rahway, NJ, 1996). For aqueous samples, serial dilutions from 0 to 1 µM digoxin were prepared using sample buffer (SB) (90 vol % 50 mM TRIS-HCl pH 7.4, 50 mM KCl, 0.1 mg/mL BSA, 20 mM EDTA, 10 vol % glycerol). Serum and urine samples were prepared by spiking with serially diluted digoxin stock solutions. Concentrations of samples prepared in-house were confirmed by comparison with two commercial digoxin standard solutions. Solid-phase extraction (SPE) was performed using the following protocol. Proteins were initially precipitated from 1 mL of urine and serum samples by addition of 200 µL of 200 mM ZnSO4, with vortexing for 1 min followed by centrifugation at 14000g for 10 min in an Eppendorf microcentrifuge (Eppendorf, Westbury, NY). A 700-µL aliquot of the supernatant was then transferred to an equilibrated Oasis HLB SPE column containing 10 mg of stationary phase. The SPE cartridges were conditioned with 1 mL of acetonitrile followed by 3 × 1 mL of water. All flow rates were ∼1 (33) Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.; Tai, M. S.; Novotny, J.; Margolies, M. N.; Ridge, R. J.; Bruccoleri, R. E.; Haber, E.; Crea, R.; et al. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5879-5883. (34) Ausubel, F. M., Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman, J. G.; Smith, J. A.; Struhl, K. Current Protocols in Molecular Biology; John Wiley & Sons: New York, 1998.

mL/min. After the sample was extracted, the cartridge was washed with 2 × 1 mL of a 1 vol % solution of acetic acid in acetonitrile/water (10/90, v/v), and 2 × 1 mL of a 1 vol % solution of ammonium hydroxide in acetonitrile/water (10/90, v/v) in order to remove acidic and basic sample matrix components. Salt was washed out with 3 × 1 mL of water, and the cartridge was then dried by applying full vacuum for 5 min. The hydrophobic sample components (including digoxin) were eluted by passing 2 × 75 µL of acetonitrile through the SPE column. The eluant was evaporated using a vacuum centrifuge (Speed-Vac concentrator, Savant Instruments, Farmingdale, NY). The dried sample was then reconstituted with 18 µL of sample buffer and analyzed by APCE as described below. APCE Analysis. An 18-µL aliquot of sample was mixed with 2 µL of scFv* stock solution (100 nM) and injected into CE analysis. Siliconized 500-µL microcentrifuge tubes were used, and the time between mixing and injection was at least 5 min. PVAcoated capillaries, 50 µm i.d., were used for all separations, with a total length of 32 cm and effective length of 25 cm, unless otherwise noted. The background electrolyte (BGE) was 50 mM CAPSO, 100 mM TRIS, pH 9, with 0.1 mg/mL BSA. Separations were conducted using the following program: Prior to each injection, the capillary was rinsed with the BGE for 1 min at 20 psi, followed by a short plug (0.2 min, 20 psi) of 10 mg/mL BSA solution in the BGE to passivate the capillary wall, and then flushed for 1 min with the BGE. The samples were pressure injected (∼0.5 psi) for 40 s on the cathode side, resulting in an injection volume of ∼66 nL. Following sample injection, a lowpressure injection (∼0.5 psi, 1 min) of BGE was applied to move the injected sample plug into the cooled region of the capillary cartridge before applying a 1-min linear voltage ramp from 0 to 20 kV, resulting in final current of 8 A. Peak areas were integrated with Beckman-Coulter System Gold software. Additional data analysis and plotting were carried out using Origin 6.0 (Microcal, Northampton, MA). RESULTS AND DISCUSSION Affinity probe capillary electrophoresis offers a general means to perform noncompetitive immunoassays but requires electrophoretically uniform reagents, and the heterogeneity of natural antibodies has been a limitation. To make a uniform reagent from an scFv, site-directed mutagenesis is first used to create a unique cysteine for a thiol-specific fluorescent label. The strong but reversible binding of His-tagged recombinant proteins to immobilized metal resins enables a rapid and general strategy for efficient labeling and purification. The applicability of APCE is demonstrated in three formats. First, a mix-and-inject method is optimized to provide low-picomolar sensitivity suitable for many clinical and biochemical applications. The use of a charged competitive ligand (shift ligand)22 in the background electrolyte offers very rapid electrophoretic resolution of analyte-bound from unbound excess affinity probe. Finally, APCE is demonstrated in combination with two solid-phase extraction methods for ultratrace sensitivity in complex samples. Figure 1A illustrates the relationship between an scFv and a native antibody. A serum antibody (immunoglobulin G, IgG), shown schematically in Figure 1A, is a Y-shaped tetramer of two heavy chains (white) and light chains (gray). Each immunoglobulin (Ig) domain (oval) is a protein-folding unit stabilized by a Analytical Chemistry, Vol. 72, No. 23, December 1, 2000

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Figure 1. Schematic diagram of preparation of uniform reagent from scFv gene. See text for details.

conserved internal disulfide bond. The antigen-binding sites are formed at the interface of the N-terminal domains (variable domains) of one heavy chain and one light chain. An scFv is a recombinant fusion protein of the light-chain variable domain (VL) with the heavy-chain variable domain (VH) into a single polypeptide chain. The domains are tethered by a flexible peptide linker, often (Gly4Ser)3 as in the present work. The fusion protein is produced by first creating a DNA construct with the VH and VL genes, such as shown in Figure 1B. As discussed above, scFv with nanomolar affinities for a desired analyte may be selected from phage display libraries in as little as two weeks.25 1. Generation of Uniform scFv Reagent. Methods for generating scFv are well established; therefore, the starting point for this investigation was an existing DNA plasmid for expressing an anti-digoxin scFv. Because even conservative amino acid changes may have unpredictable effects on scFv folding efficiency29,30 and therefore activity, four variants were made in parallel and one was selected for further study. First, separate 5782

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N-terminal and C-terminal cysteine mutant genes were made and combined with different tag sequences. The pET25 expression plasmid selected adds a 6-histidine sequence (His tag) for IMAC purification, and a 16-amino acid immunoaffinity tag (HSV tag, Novagen) (Figure 1B). The parent scFv and the four mutants were expressed in bacteria, where the protein product forms inclusion bodiessinsoluble aggregates inside the cell.28 To recover the scFv, the inclusion bodies were solubilized with urea, and the denatured scFv was purified using IMAC (Ni-NTA-agarose), as shown in Figure 1C. For initial studies, the scFv was eluted in urea and refolded by dialysis against Fv buffer.27 Exposure of either mutant or parent to 5-IAF in solution resulted in complete disappearance of the scFv (SDS-PAGE analysis), presumably due to precipitation. Aggregation of proteins during renaturation has been prevented by immobilizing the protein to a solid phase (matrix-assisted refolding).28 An extension of this strategy to matrix-assisted labeling of the scFv while immobilized on IMAC resin was explored (Figure 1D). This approach allowed excess dye to be incubated with the scFv during the labeling reaction and then removed before solubilizing the scFv with EDTA. Both the N-Cys and C-Cys mutant scFv proteins were labeled by this method, but the parent was not (data not shown). This result indicated that labeling was specific for the added cysteine and that the internal disulfide bonds (a conserved feature of immunoglobulin domains) were protected. The Nterminal variant detailed in Figure 1B was obtained in higher yield and selected for the remainder of this work (hereinafter called scFv*). The strategy of matrix-assisted labeling is in principle generally applicable to other scFv’s. Although the IMAC-purified scFv was highly uniform on denaturing SDS gels, an additional purification step was required. After labeling and elution from the IMAC column, the scFv* showed two peaks in CE-LIF analysis: In addition to the sharp peak of active protein shown below, an earlier peak was also present. The amount of active scFv* would decrease over several weeks of storage, while the early peak increased. Further purification was made by passage through a 0.02-µm filter. The filtrate contained highly uniform scFv*, which was active in APCE and stable unfrozen at -12 °C for over 1 year. Figure 2B shows an electropherogram of the scFv* labeled and purified as described above. The electropherogram is dominated by a single sharp peak, labeled scFv*. The peak X was also seen when buffer (without scFv*) was injected (Figure 2A) and so was attributed to scattering of the excitation laser by focused buffer ion fronts passing through the detector. Minor peaks (m) below 3% of the total intensity may be due to deamidation. The minor peaks did not change during prolonged storage of purified scFv. In this case, the minor peaks did not interfere with quantitation of digoxin-scFv* complex. If desired, the scFv* could be additionally purified by CE fraction collection35 or by preparative isoelectric focusing (IEF). The dissociation constant (Kd) of the labeled N-Cys scFv for digoxin, determined by titration with digoxin standard solutions, was found to be 2.5 ( 0.5 nM, in agreement with published values of the parent scFv(dig) of 1-2.2 nM.36 (35) Minarik, M.; Foret, F.; Karger, B. L. Electrophoresis 2000, 21, 247-254. (36) Daugherty, P. S.; Chen, G.; Olsen, M. J.; Iverson, B. L.; Georgiou, G. Protein Eng. 1998, 11, 825-832.

Figure 2. APCE analysis of representative digoxin concentrations. CE-LIF electropherograms of sample buffer without scFv* (A), and of 10 nM scFv* with no digoxin (B), 866 pM digoxin (C), 2.66 nM digoxin (D), and 8.66 nM digoxin (E). Electrophoretic conditions: PVAcoated capillary, 25/32 cm, 50 µm i.d., 40-s sample injection under 0.5 psi at cathode end, 1-min low-pressure buffer injection under 0.5 psi; 20 kV, 8 µA. Background electrolyte: 50 mM CAPSO, 100 mM TRIS pH 9, 0.1 mg/mL BSA. Sample electrolyte: 90 vol % 50 mM TRIS-HCl pH 7.4, 50 mM KCl, 20 mM EDTA, 0.1 mg/mL BSA, and 10 vol % glycerol. Fluorescence excitation at 488 nm and emission at 520 nm.

The overall yield was 100 µg of labeled, filtered scFv* from 10 mL of bacterial culture, sufficient for 5000-10 000 assays. The time required for mutagenesis and initial expression was ∼2 weeks. To reproduce a selected scFv, 200 mL of bacteria could be regrown from stored cultures (glycerol stocks) to produce 20 aliquots of washed inclusion bodies in 2 days. From a frozen aliquot, 1 day was sufficient for IMAC purification and fluorescent labeling, with overnight dialysis. This facile preparation of a uniform reagent enabled the following investigations of the limits of detection and speed of APCE. 2. APCE Assay of Aqueous Digoxin Standards. Figure 2 shows APCE analysis of several representative concentrations of aqueous digoxin standard solutions. For APCE analysis, only one mixing step was necessary: The scFv* was added directly to the sample, which was then injected for CE separation. In samples containing digoxin, an additional peak was observed eluting after the scFv*, with intensity directly proportional to sample digoxin concentration.

The mix-and-inject format above is the simplest implementation of APCE. Experimental parameters such as injection time, separation voltage, and capillary length were first examined in analysis of aqueous digoxin standards. To improve sensitivity, a sample stacking procedure was established using transient isotachophoresis (t-ITP).37 Sample stacking permits increasing the injected sample volume without significant band broadening. It was found experimentally that a 40-s injection at 0.5 psi in a 25/32-cm capillary gave optimum limits of detection, balancing injected sample mass with separation efficiency. PVA-coated capillaries were used to minimize possible interactions between the scFv protein and the capillary wall. Additionally, the capillaries were passivated by flushing with a short plug of 10 mg/mL BSA prior to each run, and BSA was added to the background electrolyte at 0.1 mg/mL. Run-to-run repeatability was within the variability of pressure injection, as discussed below. Precautions in sample preparation, appropriate whenever handling low-nanomolar concentrations of proteins, were the use of siliconized or BSA-passivated microtubes and minimizing contact of scFv* with untreated surfaces. The stability of an antibody-antigen complex is highly dependent on temperature. The inlet 4 cm of the capillary projects from the PACE cartridge and is not cooled. To avoid heating the sample, sample injection was followed by low-pressure injection of background electrolyte for 1 min to push the sample zone inside the cartridge before voltage was applied. When digoxin-saturated scFv* was injected without this push step, the scFv*-digoxin peak was highly variable, in the range 40-70% of the net scFv* intensity (sum of the scFv* and scFv*-digoxin peak integrals). With the push step, 85% of the injected complex was reproducibly seen at the detector. This amount of dissociation during a 10-min electrophoretic run is consistent with the published dissociation rate (koff) of the parent scFv-digoxin complex.36 As discussed previously,19,22 the ratio of the complex detected relative to that formed in the injected sample mixture can be maintained constant by keeping the separation time constant, and thus calibration will provide accurate analyte concentration values. The equilibration time necessary for the scFv*-digoxin complex to form was examined by injecting samples at various times after mixing with scFv*. Note that the low-pressure injection described above to move the injected sample into the cartridge provided a minimum equilibration time of 1 min. The intensity of the scFv*-digoxin peak was the same when samples were injected within 30 s of mixing or after several hours. This result is similar to the kinetics observed in APCE analysis of growth hormone.19 Figure 3 shows a calibration curve of APCE analysis of aqueous digoxin standards using the conditions optimized above. The lower limit of detection (LOD) was 10 pM digoxin, defined as a S/N of 3 for the scFv*-digoxin peak height. The upper limit of detection was 10 nM digoxin, limited by saturation of the added scFv* (seen as the plateau of the calibration curve). The assay showed a wide linear range of 3 orders of magnitude, r2 > 0.998 from 20 pM to 5 nM. The upper limit of detection and the linear range may be increased by adding higher concentrations of scFv*. However, at very high scFv* concentrations, trace scFv* contaminants (below 0.01%) may increase background, raising the lower limit of (37) Foret, F.; Szoko, E.; Karger, B. L. Electrophoresis 1993, 14, 417-428.

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Figure 3. Calibration curve for APCE analysis of aqueous digoxin standard solutions. Average of integrated areas of scFv*-digoxin peak in CE-LIF electropherograms as shown in Figure 2, for three repetitions of analysis at each indicated digoxin concentration.

detection. To assess the reproducibility of the assay, 15 independent samples of 433 pM digoxin were analyzed by APCE. The peak area of the scFv*-digoxin peak showed relative standard deviations below 2.5%. 3. Fast APCE Using Short Capillaries and Shift Ligand. The analysis of digoxin demonstrated above was dependent on the electrophoretic resolution of the free excess scFv* from the scFv*-digoxin complex. For some analytes, however, achieving resolution of the complex may be problematic. A general method for achieving resolution of a receptor from its complex with ligand is the addition of a charged competitive ligand (shift ligand) to the background electrolyte.22 The shift ligand interacts with the unliganded receptor, altering its mobility through added charge. The analyte-bound receptor is blocked from interaction with the shift ligand. The resulting effective mobility difference between the analyte-bound and excess receptor can dramatically increase the resolution and speed of separation. It should be noted that even when a competitive ligand is used to facilitate separation of analyte-bound antibody from free antibody, APCE is a noncompetitive assay which provides a direct and linear measurement of the number of analyte-filled antibody sites. Figure 4 shows APCE analysis of digoxin when the background electrolyte contains a digoxigenin-DNA 30-mer conjugate (digDNA) as a shift ligand. The digoxigenin moiety of the digDNA is bound by the digoxin binding site of the scFv*, and the covalently linked DNA carries a high charge. The free scFv* in the sample appears as the scFv*-digDNA peak in the electropherogram, which comigrated with the salt front. Using a 7-cm effective length/25-cm total length capillary (the shortest possible on the Beckman PACE), the scFv*-digoxin peak was detected in 1.3 min, with a lower LOD of 1 nM digoxin in aqueous standards. The limit of detection in the short capillary was significantly higher than the optimized assay above because the injected sample volume (∼10 nL) must be smaller in the short capillary. The electrophoretic efficiency is lower because the injected sample filled proportionately more of the effective capillary length, and most of the effective length was not cooled during separation. Given the resolution of the scFv*-digoxin and scFv*-digDNA peaks shown in Figure 4, APCE in capillaries or microfluidic 5784 Analytical Chemistry, Vol. 72, No. 23, December 1, 2000

Figure 4. Fast APCE: CE-LIF electropherogram of APCE in a 7-cm effective length capillary with a charged competitive ligand (shift ligand, see text) in the background electrolyte. Sample: 50 nM scFv*, 27 nM digoxin, and 1 nM fluorescein-labeled human carbonic anhydrase22 as an internal standard (Std.). Electrophoretic conditions: PVA-coated capillary, 7/25 cm, 50 µm i.d., 10-s injection under 0.5 psi at cathode end, 30 kV. Background electrolyte: 50 mM CAPSO, 100 mM TRIS pH 9, 0.1 mg/mL BSA, 20 nM digoxigenin30-mer DNA conjugate (digDNA). Sample electrolyte: 90 vol % 50 mM TRIS-HCl pH 7.4, 50 mM KCl, 0.1 mg/mL BSA, and 10 vol % glycerol. Fluorescence excitation at 488 nm and emission at 520 nm.

channels shorter than 7 cm should permit analysis of low- to midnanomolar analyte in substantially less than 1 min. 4. Solid-Phase Extraction of Aqueous Standards. The mixand-inject APCE assay described above resulted in a LOD of 10 pM digoxin in aqueous solutions. Sandwich ELISA formats, by extracting much larger sample volumes, are capable of detecting proteins below 1 pM.6 To demonstrate the applicability of APCE for ultratrace analysis, preconcentration methods were investigated. Reversed-phase solid-phase extraction (RP-SPE) is a fast and efficient cleanup and preconcentration method for biosamples, widely used in clinical applications.38 For digoxin assay, we investigated this simple off-line approach for preconcentration. One-milliliter samples were extracted using Waters Oasis HLB cartridges. The cartridges, containing 10 mg of stationary phase, were not overloaded by sample volumes up to 2 mL. Breakthrough of digoxin was not observed below 20 vol % acetonitrile. After washing, the sample was eluted with acetonitrile, dried, and then reconstituted with sample electrolyte. Increasing the preconcentration factor by using a lower volume for reconstitution was possible if samples were analyzed immediately; however, evaporation of the sample electrolyte while queued on the CE instrument introduced systematic error and limited the reproducibility of the assay if sample volumes were below 10 µL. Electropherograms of RP-SPE extracted samples were similar to those shown above for direct injection APCE assay. The calibration curve (not shown) was similarly linear over more than 3 orders of magnitude, r2 > 0.994 and an LOD of 200 fM of digoxin in a 1-mL aqueous sample. Reversed-phase extraction was wellsuited for APCE because the sample could be reconstituted in an optimal electrophoresis buffer. (38) Hennion, M. C. J. Chromatogr., A 1999, 856, 3-54.

Figure 6. CE-LIF electropherograms comparing recovery of 10.5 pM digoxin from aqueous standard solutions (A) and precipitated serum (B) using reversed-phase solid-phase extraction (text). Extracted sample volume, 700 µL. Digoxin concentration after extraction, 180 pM. Electrophoretic conditions as in Figure 2.

Figure 5. CE-LIF electropherograms showing APCE analysis of clinical digoxin concentrations, using a mix-and-inject format with 10fold sample dilution. The top two panels show analysis of Behring digoxin calibration standards at 1 ng/mL ) 1.28 nM (128 pM injected) (A) and 5 ng/mL ) 6.4 nM (640 pM injected) (B). The lower two panels show analysis of urine with no digoxin (C) and 2.56 nM digoxin (256 pM after dilution) (D). Endogenous fluorescent components of urine are noted in (C). Insets show 2.5-fold expansion of the scFv*-dig peak. Peaks X and m are described in Figure 2. Electrophoretic conditions as in Figure 2.

5. APCE Assay of Serum and Urine. The applicability of APCE in analysis of serum and urine samples was next examined. Interferences such as salts, which alter migration times, reduce stacking efficiency, or affect the off-rate of the affinity probeanalyte complex, may be reduced by initial dilution of the sample. This approach raises the lower limit of detection of the assay proportional to the amount of dilution, but is fast, is simple to implement, and ensures the longevity of the analytical column. Figure 5 shows analysis of clinical digoxin concentrations using APCE with 10-fold dilution of the sample. Even with dilution, the sensitivity would be sufficient for many applications, such as therapeutic levels of digoxin and other drugs. In analysis of serum samples, the use of 20 mM ZnSO4 to precipitate proteins did not interfere with APCE. The calibration curves for crude and precipitated serum were superimposable over their entire range from 200 pM to 200 nM before dilution (injected concentrations 20 pM to 20 nM). The limit of detection for serum samples was 200 pM before 1:10 dilution (20 pM injected). In urine samples, many fluorescent components were observed (Figure 5C,D). In this case, these interfering agents migrated with higher mobility than the scFv*-digoxin complex and did not interfere with quantitation. For general application, interferences may be resolved either chromatographically by optimizing separa-

tion conditions or spectrally by using a fluorochrome that does not overlap with interfering signals. Additionally, the mobility of an scFv reagent may be altered by using mutagenesis to add charged amino acids. Although many urine samples could be analyzed directly, a dilution factor of 1:10 gave consistent results for all collections. Calibration curves were similar to those for serum and aqueous samples. A background signal comigrating with the scFv*-digoxin complex was observed in the APCE of some urine samples not spiked with digoxin. This threshold signal was observed only in some collections of urine and was presumably caused by a matrix component that could cross-react with the scFv*, such as endogenous steroids or metabolites. This background signal limited the sensitivity of the urine assay to a LOD of 30 pM, after dilution. 6. Solid-Phase Extraction of Biological Samples. Solidphase extraction was then employed to preconcentrate digoxin and remove interfering agents from serum and urine samples. For all experiments, the protocol described above for aqueous samples was used without further modification. The two washing steps (1 vol % acetic acid and 1 vol % ammonium hydroxide) were included to remove remaining salts and peptides from the SPE cartridge. No overloading of the 10-mg HLB cartridge was seen with sample volumes up to 2 mL. Figure 6 compares electropherograms of extracted serum and aqueous samples. The electropherogram for extracted serum shows only minor additional peaks originating from crude serum. Due to the lower effective sample volume of 700 µL of supernatant after 1 mL of samples was precipitated, the lower limit of detection for serum samples was 400 fM. Calibration curves obtained for this series of experiments had the same slope as the assays above, and were linear over 3 orders of magnitude. (r2 > 0.998). Recovery of digoxin from serum and aqueous samples was equivalent, reflecting the efficiency of the SPE cleanup and preconcentration protocol. Recovery for all samples, confirmed by a series of runs, was better than 96%, and reproducibility of the APCE analysis, as described above, was within 2.5%. As an alternative to reversed-phase SPE as demonstrated above, immunoaffinity extraction of digoxin using the scFv* Analytical Chemistry, Vol. 72, No. 23, December 1, 2000

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immobilized on IMAC resin (Fv-IMAC) was also possible (data not shown). After the sample was extracted and the resin washed, the scFv* was eluted under native conditions with sample electrolyte containing 20 mM EDTA and injected directly into APCE analysis. Serum and urine samples required preclearing over Ni-IMAC before digoxin extraction over Fv-IMAC. Immunoaffinity extraction may be more applicable to labile protein analytes which would be denatured by organic solvents used in reversed-phase extraction. Furthermore, because it does not require solvent exchange, this technique may be implemented for in-line sample preconcentration in capillaries or microfluidic devices. CONCLUSION The above results demonstrate the development of a rapid and sensitive assay, using an scFv specific for digoxin as a model system. Methods are well-established for producing new scFv’s, with specificity for any desired analyte.25 With scFv as a source of uniform immunoreagents, APCE should be a generally applicable method for noncompetitive immunoassay of small analytes, filling an unmet need in available immunoassay technology. As a general approach, mutagenesis and uniform labeling of a new scFv could require effort comparable to obtaining antibodies. However, in addition to the greater sensitivity and linear range of APCE as a noncompetitive format, and the higher specificity and reproducibility of a monoclonal reagent, recombinant expression systems offer long-term advantages. The mobility of the scFv may be adjusted by using mutagenesis to add or remove charged amino acids. Any existing scFv may be regrown rapidly from frozen bacterial cultures, and the purification and labeling may be scaled up to economically supply large quantities of reagents.

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The low reagent consumption (a 10-mL bacterial culture supplied enough reagent for 10 000 assays) may be advantageous for highthroughput screening applications. CE immunoassays have shown good performance on microfabricated devices,17,39 and CE has gained acceptance in clinical assay applications.40 APCE’s combination of simplicity, sensitivity, and rapid turnaround time would permit clinical assay of a wide range of analytes on a single platform. APCE may also detect multiple analytes simultaneously, using two or more affinity probes with different mobilities. Abbreviations: scFv, single-chain antibody variable-region fragment; Ab, antibody; Fab, antibody-antigen binding fragment; hCAII, human carbonic anhydrase II; CE-IA, capillary electrophoretic immunoassay; CZE, capillary zone electrophoresis; APCE, affinity probe capillary electrophoresis; IEF, isoelectric focusing; LIF, laser-induced fluorescence; IMAC, immobilized metal affinity chromatography; NTA, nitrilotriacetic acid; SPE, solid-phase extraction; PVA, poly(vinyl alcohol); IAF, iodoacetamidofluorescein; digDNA, digoxigenin-DNA 30-mer conjugate; BGE, background electrolyte; BSA, bovine serum albumin. ACKNOWLEDGMENT This work was supported by NIH Grant GM15847. This paper is contribution number 784 from the Barnett Institute. Received for review July 27, 2000. Accepted September 19, 2000. AC000853+ (39) Schmalzing, D.; Koutny, L. B.; Taylor, T. A.; Nashabeh, W.; Fuchs, M. J. Chromatogr., B 1997, 697, 175-180. (40) Jellum, E.; Dollekamp, H.; Brunsvig, A.; Gislefoss, R. J Chromatogr., B 1997, 689, 155-164.