Anal. Chem. 2001, 73, 1134-1139
A Genetically Engineered Fusion Protein with Horseradish Peroxidase as a Marker Enzyme for Use in Competitive Immunoassays Vitaly Grigorenko,† Irina Andreeva,† Torsten Bo 1 rchers,* Friedrich Spener,§ and Alexey Egorov†,‡
Institut fu¨r Chemo und Biosensorik, Mendelstrasse 7, D-48149 Mu¨nster, Germany
Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making. Enzyme immunoassays for detection and quantitative analysis of various substances are based on the coupling of marker enzymes such as horseradish peroxidase (HRP) with antigens or antibodies. Two main approaches are used for chemical conjugation of proteins and haptens to peroxidase, coupling with glutaraldehyde,1 or making use of the oligosaccharide component of the enzyme by oxidation of the enzyme with sodium m-periodate.2 Another means for production of conjugates is based on modification of thiol groups with N,N′-o-phenylenedimaleimide. Antigens with free SH- groups can then be directly coupled to the modified enzyme.3 However, all of these methods result in partial inactiva* Corresponding author. Phone: +49-251-980 2880. Fax: +49-251-980 2890. E-mail:
[email protected]. † Immunotek of Moscow State University, Moscow, Russia. ‡ Laboratory of Enzyme Engineering, Chemistry Department of Moscow State University. § Department of Biochemistry, University of Mu ¨ nster, Mu ¨ nster, Germany. (1) Avrameas, S. Immunochemistry 1969, 6, 43-52. (2) Nakane P. K.; Kawaoi, A. J. Histochem. Cytochem. 1974, 22, 1084-1091. (3) Kato, K.; Hamaguchi, Y.; Fukui, H.; Ishikawa, E. J. Biochem. 1975, 78, 235237.
1134 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
tion of the enzyme and heterogeneity of the conjugates, which in turn influence specificity and sensitivity of the immunoassays.4,5 With advances in genetic engineering, it became clear that genetic in-frame fusions of antigens and enzymes would provide many of the desirable features of conjugates for use in immunoassays, in particular homogeneity, 1:1 stoichiometry, reproducibility, and ease of production.4 Early fusion proteins contained the bacterial enzymes β-galactosidase6-8 or alkaline phosphatase,9-11 which can be easily expressed in Escherichia coli. In addition to these enzymes, bioluminescent or fluorescent marker proteins such as aequorin or green fluorescent protein12,13 have been used as fusion partners for a model octapeptide. Genetic fusion was also employed to construct conjugates with protein A14 or an in vitro biotinylated polypeptide tag for β-galactosidase.15 The genetic approach is particularly attractive for fusions to small peptides with numerous functional groups, which are difficult to control,16 or with human proteins which often are not easily available.17 Whereas β-galactosidase is solubly expressed in the cytosplasm, the disulfide-containing alkaline phosphatase is secreted into the periplasm, thus also broadening the spectrum of fusions to disulfide-containing proteins.18,19 The drawback of the lower specific activity of the bacterial alkaline phosphatase in comparison to the calf intestinal alkaline phosphatase routinely used in (4) Lindbladh, C.; Mosbach, K.; Bulow, L. Trends Biochem. Sci. 1993, 8, 279283. (5) Porstman. T.; Kiessig, S. T. J. Immunol. Methods 1992, 150, 5-21. (6) Offensperger, W.; Wahl, S.; Neurath, A. R.; Price, P.; Strick, N.; Kent, S. B.; Christman, J. K.; Acs, G. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 75407544. (7) Peterhans, A.; Mecklenburg, M.; Meussdoerffer, F.; Mosbach, K. Anal. Biochem. 1987, 163, 470-475. (8) Markaryan, A. N.; Mashko, S. V.; Kukel, L. V.; Lapidus, A. L.; Bach, A. N.; Egorov, A. M. Ann. N. Y. Acad. Sci. 1991, 646, 125-135. (9) Lindbladh, C.; Persson, M.; Bu ¨ low, L.; Stahl, S.; Mosbach, K. Biochem. Biophys. Res. Commun. 1987, 149, 607-614. (10) Gillet, D.; Ezan, E.; Ducancel, F.; Gaillard, C.; Ardouin, T.; Istin, M.; Me´nez, A.; Boulain, J.-C.; Grogent, J.-M. Anal. Chem. 1993, 65, 1779-1784. (11) Ezan, E.; Ducancel. F.; Gillet, D.; Drevet, P.; Me´nez, A.; Grognet, J. M.; Boulain, J. C. J. Immunol. Methods 1994, 169, 205-211. (12) Ramanathan, S.; Lewis, J. C.; Kindy, M. S.; Daunert, S. Anal. Chim. Acta 1998, 369, 181-188. (13) Lewis, J. C.; Daunert, S. Anal. Chem. 1999, 71, 4321-4327. (14) Lindbladh, C.; Mosbach, K.; Bu ¨low, L. J. Immunol. Methods 1991, 137, 199207. (15) Wittkowski, A.; Kindy, M. S.; Daunert, S.; Bachas; L. G. Anal. Chem. 1995, 67, 1301-1306. (16) Wittkowski, A.; Daunert, S.; Kindy, M. S.; Bachas, L. G. Anal. Chem. 1993, 65, 1147-1151. (17) Schreiber, A.; Specht, B.; Pelsers, M. M. A. L.; Glatz, J. F. C.; Bo¨rchers, T.; Spener, F. Clin. Chem. Lab. Med. 1998, 36, 283-288. 10.1021/ac000684t CCC: $20.00
© 2001 American Chemical Society Published on Web 02/14/2001
chemical conjugations could be partly overcome by using a genetically engineered mutant of the bacterial enzyme with increased activity.20 A principal problem associated with β-galactosidase or alkaline phosphatase fusions is their tetrameric and dimeric structure, respectively, which likely leads to an increased apparent affinity (avidity) of a conjugate in comparison to the free antigen. This is not desirable for the development of competitive immunoassays. On the other hand, horseradish peroxidase, which is very popular for preparation of enzyme-conjugates,5 can only be expressed in E. coli in the form of inclusion bodies. The yield of refolded and reconstituted (with heme) recombinant peroxidase used to be rather low,21,22 which has so far precluded the use of this enzyme in the genetic fusion approach. The recent progress in heterologous expression in E. coli and reactivation of recombinant HRP carrying a C-terminal oligo-histidine tag (HRPhis)23 opened the prospect of producing a recombinant conjugate of HRPhis with a marker enzyme for application in immunoassays. In the present paper, we demonstrate that a fusion protein of peroxidase and human heart fatty acid binding protein (H-FABP) can be used as a recombinant tracer (HRP-FABPhis) in immunoassays for the detection of H-FABP. This small (15 kDa) cytosolic protein is a member of a protein family that specializes in the transport of fatty acids24 and is highly abundant in heart muscle25 (0.52 mg/g heart tissue). Its rapid release into the circulation and its good tissue specificity make it an ideal early marker for clinical diagnosis of acute myocardial infarction.26,27 In recent clinical studies, H-FABP was determined by sandwich ELISA.26 To support the clinical decision-making process, it is desirable that the assay time be as short as possible. This stimulated the development of an electrochemical immunosensor.28 With prefabricated, screen-printed electrodes, an assay time of 20 min with a measuring range of 10-350 ng/mL was achieved, covering the pathological range of H-FABP in the circulation and exhibiting good correlation with the reference sandwich ELISA.29 Here we demonstrate that the competitive assay format with its reduced number of incubations can also be employed to develop fast immunoassays, even in the popular microtiter format. (18) Gillet, D.; Ducancel, F.; Pradel, E.; Le´onetti, M.; Me´nez, A.; Boulain, J. C. Protein Eng. 1992, 5, 273-278. (19) Chanussot, C.; Bellanger, L.; Ligny-Lemaire, C.; Seguin, P.; Me´nez, A.; Boulain, J. C. J. Immunol. Methods 1996, 197, 39-49. (20) Kerschbaumer, R. J.; Hirschl, S.; Schwager, C.; Ibl, M.; Himmler, G. Immunotechnology 1996, 2, 145-150. (21) Smith, A. T.; Santana, N.; Dacey, S.; Edwards, M.; Bray, R. C.; Thorneley, R. N. F.; Burke, J. F. J. Biol. Chem. 1990, 265, 13335-13343. (22) Egorov, A. M.; Gazaryan I. G.; Kim, B. B.; Doseeva, V. V.; Kapeliuch, J. L.; Veryovkin, A. N.; Fechina, V. A. Ann. N. Y. Acad. Sci. 1994, 721, 73-82. (23) Grigorenko, V.; Chubar, T.; Kapeliuch, Yu.; Bo¨rchers, T.; Spener, F.; Egorov, A. Biocatal. Biotransform. 1999, 17, 359-397. (24) Bo ¨rchers, T.; Spener, F. Curr. Top. Membr. 1994, 40, 261-294. (25) Van Nieuwenhoven, F. A.; Kleine, A. H.; Wodzig, K. W. H.; Hermens, W. T.; Kragten, H. A.; Maessen, J. G.; Punt, C. D.; Van Dieijen, M. P.; van der Vusse, G. J.; Glatz, J. F. C. Circulation 1995, 92, 2848-2854 (26) Wodzig, K. W. H.; Pelsers, M. M. A. L.; van der Vusse, G. J.; Roos, W.; Glatz, J. F. C. Ann. Clin. Biochem. 1997, 34, 263-268. (27) Glatz, J. F. C.; van der Vusse, G. J.; Simoons, M. L.; Kragten, J. A.; van Dieijen-Visser, M. P.; Hermens, W. T. Clin. Chim. Acta 1998, 272, 87-92. (28) Schreiber, A.; Feldbru ¨ gge, R.; Key, G.; Glatz, J. F. C.; Spener, F. Biosens. Bioelectron. 1997, 12, 1131-1137. (29) Key, G.; Schreiber, A.; Feldbru ¨ gge, R.; McNeil, C. J.; Jørgensen, P.; Pelsers, M. M. A. L.; Glatz, J. F. C.; Spener, F. Clin. Biochem. 1999, 32, 229-231.
EXPERIMENTAL SECTION Reagents. 2,2-Azino-bis(3-ethyl-thiazoline-6-sulfonate) diammonium salt (ABTS), phenol and 4-aminoantipyrine, isopropyl-βD-thiogalactopyranoside (IPTG), sodium dodecyl sulfate (SDS), Tris, glycerol, oxidized glutathione, dithiothreitol (DDT), calcium chloride, hemin, o-phenylendiamine (OPD tablets), and other components of buffer and refolding solutions were purchased from Sigma (Deisenhofen, Germany). Bactotryptone and yeast extract were purchased from Difco (Augsburg, Germany). Restriction endonucleases, ligase, and other DNA modifying enzymes and kits were molecular biology grade and purchased from various suppliers. Oligonucleotide primers for PCR were synthesized by MWG-Biotech (Ebersberg, Germany). Recombinant human H-FABP for calibration was produced in E. coli as previously described.17 Monoclonal antibodies 67D3 and 66E2 were kindly provided by Dr. Jan F. C. Glatz (Maastricht, The Netherlands). Heparinized plasma of a patient enrolled in the Eurocardi multicenter study30 and earlier analyzed by sandwich ELISA26 and immunosensor29 was used for evaluation of the competitive immunoassay. Cloning Strategy. A fusion protein comprising HRP and human H-FABP was constructed by inserting the coding part of the human H-FABP cDNA into the XmaIII site of the expression vector for horseradish peroxidase, pETHRPHis,23 just in front of the 6× His tag. For this, a PCR product with flanking XmaIII sites was generated from plasmid pCRIIhH-FABP17 using forward primer 5′-TTCGGCCGCAGGAGGATCAGTGGACGCTTTCCTGGGCACC-3′ and reverse primer 5′-TTCGGCCGCCTCTTTCTCATAAGTGCGAGTGC-3′ (XmaIII sites underlined). The amplified DNA was cloned into pGEMTeasy (Promega, Mannheim, Germany) and then subcloned into XmaIII restricted pETHRPHis, yielding pETHRP-FABPHis. The forward primer also encoded a short linker peptide (nucleotides shown in italics). Screening for clones with the correct orientation of the insert was performed by restriction analysis using PstI as well as Western blotting with a polyclonal antiserum raised against recombinant human HFABP. The correct sequence of the construct was verified by DNA sequencing (ABI BigDye cycle sequencing kit, PE Biosystems, Weiterstadt, Germany). Expression, Refolding, and Purification of Recombinant HRP-FABPhis Conjugate. E. coli BL21(DE3)pLysS bacteria were transformed with pETHRP-FABPhis (Figure 1) and grown under vigorous shaking at 37 °C in 300 mL LB medium containing 100 µg/mL ampicillin, 34 µg/mL chloramphenicol to an OD550 of 0.6 and induced with 0.4 mM IPTG. Bacteria were further grown at 30 °C for 3 h, harvested by centrifugation (3600g, 15 min, 4 °C) and lysed by freezing at -70 °C, thawing in 10 mL of buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 10 mM DTT), and further incubating on ice for 30 min. Lysis was completed by sonication with three 30-s pulses (Branson sonifier). The pellet obtained after centrifugation (10 000g, 30 min, 4 °C) was resuspended in 10 mL of buffer A, supplemented with 1% Triton X-100. The washed inclusion bodies were then solubilized in 5 mL of buffer (50 mM Tris-HCl, 6 M urea, pH 8.0, and 2 mM DTT) under continuous stirring (1 h, room temperature). Remaining insoluble (30) Glatz, J. F. C.; Haastrup, B.; Hermens, W. T.; Zwaan, C. D., Barker, J., McNeil, C.; Luscher, M.; Ravkilde, J.; Thygesen, K.; Kristensen, S.; Horder, M. Circulation 1997, 96 (suppl), I-215.
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Figure 1. Scheme for construction of pETHRP-FABPhis.
material was removed by centrifugation (10 000g, 30 min, room temperature). The solubilized inclusion bodies (5 mL) were added drop-bydrop to 95 mL of refolding medium (20 mM Tris-HCl, 1.7 M urea, 4% glycerol, pH 8.5, and 2 mM CaCl2). Thereafter, oxidized L-glutathione was added to a final concentration of 0.7 mM, and the mixture was left overnight at 4 °C without stirring. The solution now containing refolded apoHRP-FABPhis was stirred with 5 mL Ni-NTA agarose (Qiagen, Hilden, Germany) for 2 h at 4 °C. The Ni-NTA agarose was collected in a column (1.5 cm diameter) and bound apoHRP-FABPhis was eluted at room temperature using an imidazole-containing buffer (10 mM Tris-HCl, 0.15 M NaCl, 2 M urea, 200 mM imidazole, pH 7.4). Fractions containing apoHRPFABPhis were collected, dialyzed against PBS (10 mM Naphosphate, 0.15 M NaCl, pH 7.4) and reconstituted with hemin, which was added drop-by-drop from an ∼1 mM stock solution in 0.01 M KOH. The final concentration of the added hemin did not exceed 3 times the concentration of apoHRP-FABPhis, as estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The sample was concentrated by ultrafiltration and passed through a column with Sephadex G-25 (1.5 × 25 cm) equilibrated with PBS to remove unbound hemin. Enzyme Activity Measurements. For determination of the concentration of holoHRP-FABPhis, the molar absorption coefficient for peroxidase at 403 nm of 102 000 M-1 cm-1 was used.31 Peroxidase activity measurements were performed using a Shimadzu UV 1202 spectrophotometer at 25 °C according to the following protocol: 0.05 mL ABTS solution (15 mM in H2O) and an enzyme aliquot (5-20 µL) were added to 2 mL of 0.1 M sodium (31) Dunford, H. B.; Stillman, J. S. Coord. Chem. Rev. 1976, 19, 187-251.
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Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
acetate buffer, pH 5.0. The reaction was initiated by the addition of 0.1 mL of 0.5% hydrogen peroxide solution, and product formation was monitored at 405 nm using 405 ) 36 800 M-1 cm-1. The specific activity was expressed in U (µmol/min)/mg of protein. Preparation of a Chemical Conjugate of HRP and Human H-FABP. A conjugate of plant peroxidase and recombinant human H-FABP has been synthesized on the basis of periodate oxidation of oligosaccharides.2 Plant peroxidase was dissolved in 1.1 mL of 1 mM sodium acetate buffer, pH 4.4. The exact concentration was determined spectrophotometrically to be 4.82 mg/mL. Under continuous stirring, 0.2 mL of 0.2 M NaIO4 was added dropwise to the peroxidase solution in the dark and further incubated for 20 min. After extensive dialysis against 1 mM sodium acetate, pH 4.4, 0.8 mL of a solution of recombinant human H-FABP (0.98 mg/mL in 0.01 M sodium carbonate, pH 9.6) was added. After 2 h stirring at room temperature, 0.2 mL of fresh NaBH4 (4 mg/mL) was added and the mixture was incubated for an additional 30 min. The sample was extensively dialyzed against PBS and stored at -20 °C in the presence of 50% (v/v) glycerol. Of this preparation, 1 mL was passed over a Sephadex G50 column (1.6 × 50 cm) equilibrated with PBS to separate the conjugate from free H-FABP. Peroxidase-containing fractions were pooled and stored in 50% glycerol at -20 °C. The concentration of the H-FABP part in the mixture of the chemical conjugate was estimated by sandwich ELISA (see below) to be 6.3 µg/mL, which corresponds to 25 µg/mL HRP/H-FABP conjugate. The concentration of the total peroxidase estimated by the Soret band (403 nm) as 63 µg/mL is compatible with the observation from SDSPAGE that considerable amounts of free peroxidase (45 kDa) are found in addition to the conjugate with 1:1 stoichiometry and some higher molecular mass conjugate species with more than one H-FABP per peroxidase molecule. The specific peroxidase activity toward ABTS was 300 U/mg, about 30% of the starting activity. Gel Electrophoresis and Western Blot Analysis. SDSPAGE was performed in 12% gels. For Western blotting, samples were subjected to SDS-PAGE and transferred (1 h) to nitrocellulose (0.45 mm, Schleicher & Schuell, Dassel, Germany) in 25 mM Tris-base, 0.192 M glycine, 20% methanol at 2 mA/cm2 under cooling with tap water. Blots were baked at 65 °C for 1 h, blocked with 0.5% BSA in PBS, and incubated with polyclonal antiserum against human H-FABP (1:200). Bound antibodies were revealed with protein A-peroxidase, 1:3000 (Biorad, Mu¨nchen, Germany). Immunoassays. Microtiter plates (Nunc maxisorp) were coated overnight at 4 °C with affinity-purified polyclonal antibodies (2 µg/mL) that were raised against recombinant human H-FABP17 and dissolved in coating buffer (10 mM Na-carbonate, pH 9.6, 100 µL/well). Plates were subsequently washed 3 times with 150 µL of PBS-T (PBS supplemented with 0.1% (v/v) Tween-20) per well. The HRP-FABPhis conjugate and recombinant human H-FABP for calibration were diluted in PBS-T with 0.5% (w/v BSA), and 50 µL of each was pipetted per well. In some experiments, plasma from a pool of healthy subjects, which was virtually H-FABP-free, was added to the sample buffer that was used for dilution of standards and samples. Plates were incubated for 1 h at 37 °C and wells were then washed 3 times with 150 µL of PBS-T. For color reaction, 100 µL of substrate solution (0.05 M sodium citrate, pH 5.0, 0.42 mg/mL o-phenylendiamine, 0.03% (v/v) H2O2, freshly
prepared) was added, and the color signal was developed for 20 min in the dark at room temperature. The reaction was stopped by the addition of 50 µL of 2 M H2SO4. Absorption at 490 nm was determined by a microtiterplate reader (Dynatech MR5000, Denkendorf, Germany). Results are expressed in terms of B/B0, where B and B0 represent the absorbance in the presence and absence of H-FABP, respectively. The limit of detection was calculated from B ) B0 - 3 SD of B0 (three independent competition curves with 3 wells for each B0 determination). We defined the beginning of the measuring range by B ) 0.9 B0. The reference method for human H-FABP determinations was a sandwich ELISA having a linear range from 1 to 15 ng/mL, described earlier,17,26 and using the monoclonal capture antibody 67D3 and the alkaline phosphatase conjugate of the monoclonal antibody 66E2.28 For comparison, plasma samples were also analyzed by a recently developed electrochemical immunosensor28 based on the same monoclonal antibodies. Surface plasmon resonance measurements with these antibodies were kindly performed by Dr. Bernfried Specht (Inventus BioTec, Mu¨nster, Germany) using the BIACORE2000 device (BIAcore AB, Freiburg, Germany) as described earlier.28,32
Figure 2. Progress of purification of recombinant fusion protein. SDS-PAGE (12%) with Coomassie staining. Lane 1, low-molecularweight marker proteins; lane 2, HRPhis inclusion bodies; lane 3, HRPhis purified on Ni-NTA agarose and reconstituted with hemin;23 lanes 4 and 5, cell lysate of E. coli BL21(DE3)pLysS/pETHRPFABPhis before and after induction with IPTG; lane 6, HRP-FABPhis inclusion bodies solubilized in 6 M urea; lane 7, HRP-FABPhis purified on Ni-NTA agarose and reconstituted with hemin.
RESULTS AND DISCUSSION HRP is a rather complex enzyme, characterized by four disulfide bonds, the prosthetic heme group, a high content of carbohydrates, and two bound calcium ions. Consequently, the recombinant enzyme is obtained in an insoluble form in the E. coli cytoplasm.21,22 Nevertheless, we recently succeeded in optimizing a refolding procedure taking advantage of a 6× histidine tag which we introduced at the C-terminus and which allowed for gentle purification of the recombinant enzyme by metalchelating chromatography, even from the dilute solutions obtained after refolding.23 Here we describe the preparation of a fusion of peroxidase (the marker enzyme) and human heart-type fatty acid binding protein (the antigen to be analyzed) by genetic engineering. Using the pETHRP-FABPhis expression vector in which the coding part of human H-FABP is fused in the correct reading frame behind the peroxidase codons and in front of the His tag (Figure 1), the recombinant HRP-FABPhis conjugate has been overexpressed (approximately 30% of total protein) in E. coli BL21(DE3)pLysS in the form of inclusion bodies. Both of the domains of the recombinant fusion protein are held together by means of a short flexible peptide linker (Ala-Ala-Ala-Gly-Gly-Ser) which had been introduced by means of one of the PCR primers. The inclusion body pellet was solubilized in 6 M urea, yielding unfolded HRP-FABPhis of about 80% purity according to SDSPAGE (Figure 2). Refolding was carried out by dilution in the presence of 2 M urea, 2 mM CaCl2, and upon addition of oxidized glutathione by formation of disulfide bonds overnight at +4 °C. Binding of the diluted, refolded apo form of HRP-FABPhis on NiNTA agarose provided concentration and purification in one step prior to dialysis for removal of residual urea and imidazol and reconstitution with hemin. After the final gel filtration of the recombinant conjugate on Sephadex G25 to remove excess hemin, a specific peroxidase activity toward ABTS of 650 U/mg was determined. Taking into account the molecular mass difference between the recombinant conjugate (48 kDa) and HRPhis (34 kDa), this value corresponds to 920 U/mg of the HRP part, which is close to the specific activity that is observed for recombinant
HRPhis alone (1150 U/mg) and the plant-derived enzyme (1000 U/mg).23 Using this renaturation and purification protocol previously developed for recombinant HRPhis,23 a fully active recombinant HRP-FABPhis conjugate was obtained in a yield of about 4 mg from 300 mL of E. coli culture medium. Recombinant HRP-FABPhis conjugate has the same Soret band absorption with a maximum at 403 nm as does native peroxidase and recombinant HRPhis, indicating that the Fe3+ coordination by heme as well as proximal and distal histidines is not affected.23 These data indicate that C-terminal extension of the recombinant peroxidase with the 15 kDa human H-FABP has no drastic influence on the activity of the recombinant conjugate. At the same time, the recombinant conjugate bound fatty acids, as shown qualitatively by a gel elution assay with radioactive oleic acid (data not shown), and was recognized by a sandwich ELISA with two monoclonal antibodies,17 indicating the structural integrity of the FABP part. On the basis of ELISA measurements with recombinant human H-FABP as the standard, for instance, the concentration of a HRP-FABPhis fusion protein preparation was estimated to be 106 µg/mL. At the same time, estimation of the peroxidase part of this preparation by the Soret band absorption at 403 nm yielded a fusion protein concentration of 145 µg/mL. This difference may be caused by a slightly worsened recognition, for steric reasons, of the FABP part in the fusion protein by the two monoclonal antibodies. This was studied in more detail by BIAcore analysis of the binding of H-FABP and the fusion protein to both monoclonal antibodies (Table 1). The individual differences in the kinetic parameters led to a 3-fold lower affinity of the detector antibody and a 2-fold higher affinity of the capture antibody to the fusion protein and may cause the different responses of H-FABP and the fusion protein in the sandwich ELISA. The ratio of the absorbencies at 403 and 280 nm (RZ value), which is frequently taken as a measure for peroxidase purity, was slightly decreased in the fusion protein as compared to recombinant HRP. This difference reflects the increase (about 30%) of the protein absorbance at 280 nm due to the additional H-FABP Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
1137
Table 1. Kinetic Parameters for the Binding of Monoclonal Antibodies to the H-FABP Fusion Protein mAb 66E2
mAb 67D3
antigen
kon × 10-5 [M-1 s-1]
koff × 103 [s-1]
KD [nM]
kon × 10-5 [M-1 s-1]
koff × 103 [s-1]
KD [nM]
rec. human H-FABP
5.0 ( 0.3 (n ) 10)
1.9 ( 0.1 (n ) 19)
3.9 ( 0.5
3.0 ( 0.3 (n ) 21)
4.2 ( 0.3 (n ) 21)
14.2 ( 2.6
0.24 ( 0.02 (n ) 12)
0.3 ( 0.07 (n ) 12)
12.1 ( 4.3
0.8 ( 0.05 (n ) 13)
0.5 ( 0.1 (n ) 15)
6.2 ( 1.7
HRP-FABPhis
Data were determined by BIAcore analysis and are presented as mean ( SD for the number of measurements given in parentheses. The equilibrium constant for the dissociation reaction, KD, was calculated from the kinetic constants: KD ) koff /kon.
Figure 3. Titration of polyclonal anti human H-FABP antibodies (200 ng/well) with recombinant HRP-FABPhis conjugate (-b-). For the control, the recombinant conjugate was also added to polyclonal antihuman I-FABP antibodies (200 ng/well, -O-). Data are expressed as mean (n ) 2) and deviation from mean.
in the fusion protein. In the refolding step, we constantly observed a reduced precipitate formation in the case of the fusion protein in comparison to HRPhis alone. It appears that recombinant HRPFABPhis is more soluble in the absence of urea than recombinant HRPhis23 or deglycosylated HRP.33 To establish a competitive ELISA, we first studied the binding of the HRP-FABPhis conjugate to affinity-purified polyclonal rabbit antibodies (capture antibodies) raised against human H-FABP. The recombinant fusion protein bound to the capture antibodies, whereas no crossreactivity toward polyclonal antibodies raised against the homologous intestinal-type FABP (I-FABP) was observed (Figure 3). Good signals were obtained already with 2.5 ng/well of the peroxidase conjugate. At this fixed concentration, the signal obtained by oxidation of the colorimetric substrate o-phenylendiamine was linearly dependent on the amount of immobilized polyclonal anti-H-FABP antibodies up to at least 200 ng/well (data not shown). For the competition of the recombinant conjugate with free H-FABP present in standards or samples, the sequence in which the reagents were added to the wells was crucial. Once the conjugate was bound to the plate, it was not possible under the conditions applied to displace significant amounts of it, even after adding a large excess of free H-FABP (Figure 4 A). H-FABP and conjugate, however, efficiently competed for binding to the capture antiboies when added together. A slightly higher portion of H-FABP bound when wells were preincubated with H-FABP before (32) Karlsson, R.; Michaelsson, A.; Mattsson, L. J. Immunol. Methods 1991, 145, 229-240. (33) Tams, J. W.; Welinder, K. G. Anal. Biochem. 1995, 228, 48-55.
1138 Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
Figure 4. Calibration curves for the competitive immunoassay. Immobilized polyclonal anti-H-FABP antibodies (200 ng/well). A, dependence on the incubation scheme: preincubation (30 min, 37 °C) of immobilized antibodies with either recombinant conjugate (2.5 ng/well) (-9-) or H-FABP (-2-) prior to incubation (30 min, 37 °C) with the other reaction partner; co-incubation (60 min, 37 °C) of recombinant conjugate and H-FABP (-b-). B, competition of 2.5 ng/well of recombinant (-b-) or chemically prepared conjugate (-9-) with HFABP. Data are expressed as mean (n ) 2) and deviation from mean and were fitted with a 4-parameter logistic plot: y ) y0 + a/(1 + (x/x0)b) with y ) B/B0 and x ) [H-FABP].
the conjugate was added. For later experiments, the co-incubation mode was chosen. It has been recently demonstrated that it is possible to develop an online flow displacement immunoassay if from a panel of monoclonal antibodies, those with suitable kinetic binding parameters (in particular, koff, see below) can be selected.34 The typical calibration curves shown in Figure 4 B demonstrate the wide measuring range (2 orders of magnitude of H-FABP concentration) and a low detection limit of 0.1 ng/well. The intraassay variation coefficient with the recombinant conjugate was between 4 and 8% for different H-FABP concentrations. The detection limit of this new immunoassay is similar to that observed with sandwich-type ELISAs using monoclonal antibodies against human H-FABP (0.02-0.1 ng/well).17,26 Interestingly, at higher concentrations, H-FABP competed better with the recombinant conjugate than with the chemically prepared conjugate for binding to the polyclonal capture antibodies. One explanation could be the presence of chemically prepared conjugate consisting of HRP with more than one H-FABP attached, which would exhibit a higher apparent affinity due to multipoint binding to the immobilized antibodies. Preliminary experiments to establish a competitive immunoassay with the monoclonal antibody 67D3 revealed a less effective competition (data not shown), which may be rationalized by the (34) Kaptein, W. A.; Korf, J.; Cheng, S.; Yang, M.; Glatz, J. F. C.; Renneberg, R. J. Immunol. Methods 1998, 217, 103-111.
Figure 5. Competitive ELISA for H-FABP in the presence of plasma. Immobilized polyclonal anti-H-FABP antibodies (200 ng/well). A: coincubation of 50 µL of recombinant conjugate (2.5 ng/well) and a 50µL sample containing 0 (-b-) or 11 (-O-) ng/mL (0.55 ng/well) H-FABP in increasing amounts of control plasma. B: calibration curves prepared in buffer (-b-) or buffer containing 20% control plasma (-O-). Data (n ) 3) were fitted as in Figure 4.
much lower koff value observed for the fusion protein in comparison to the free H-FABP (Table 1). Presumably, amino acid residues of antibody 67D3, upon binding to H-FABP, are in contact with surface residues of the peroxidase part of the fusion protein. This steric effect may on one hand be the reason for the lower kon value observed for the binding of antibody 67D3 to the fusion protein, in comparison to H-FABP alone. Once bound, the interaction with the peroxidase part may, on the other hand, lead to sticking of the fusion protein to the capture antibody. With polyclonal antibodies, which are directed against all possible epitopes, this effect is much less pronounced. To address possible matrix effects on the competitive ELISA, the plasma dependence of the binding of the recombinant conjugate to the capture antibody in the presence and absence of 0.55 ng/well H-FABP was studied (Figure 5). As was expected, in both cases, the signal decreased slightly with increasing plasma concentrations, indicating some interference of the plasma. This would correspond to an overestimation of the H-FABP concentration in plasma, as compared to that measured using a reference sandwich ELISA, which revealed no significant matrix effect at the sample dilutions normally used. However, after an initially stronger decrease with 20% (v/v) plasma present, further addition of plasma led only to a minor additional decrease of the signal in the competitive ELISA (Figure 5A). We, thus, decided to routinely include 20% (v/v) of a virtually H-FABP-free plasma pool from healthy subjects in our incubations with the calibration samples for conditioning. The calibration curves obtained in the absence and presence of 20% “normal” plasma are compared in Figure 5B. The limit of detection of the final assay was 0.075 ng/well, which corresponds to 3.75 ng H-FABP/mL of plasma when using a 20µL sample. To test the competitive ELISA under realistic conditions, we analyzed a set of plasma samples that were periodically withdrawn from one patient, who had a diagnosis of AMI, over a period of 24 h after admission to the hospital. The H-FABP calibrators were prepared in buffer containing 20% normal plasma, as discussed above. The values for H-FABP concentrations in the plasma samples assayed with the competitive ELISA exhibited good correlation with those obtained by the reference sandwich ELISA and the recently developed EUROCARDI immunosensor26,28 (Figure 6). The measuring range from 0.2 to 30 ng/well (Figures
Figure 6. Competitive ELISA with plasma from a patient with acute myocardial infarction. Plasma (20 µL) was analyzed by competitive immunoassay (-0-) using 200 ng/well polyclonal antibodies and coincubation with 2.5 ng/well of recombinant conjugate. Data are compared to those obtained earlier by sandwich ELISA (-9-) and electrochemical immunosensor (-b-) using the same plasma samples.
4B and 5B) corresponds to 10-1500 ng of H-FABP/mL of plasma, when 20-µL samples are analyzed. If necessary, the limit of detection and the measuring range can be further improved by increasing the sample volume up to 50 µL (Figure 5A). The competitive assay is, thus, well-suited to determining not only the high concentrations of human H-FABP found in plasma of patients with acute myocardial infarction (up to few hundred ng/mL), but also H-FABP levels above the recently established discriminator value of 5 ng/mL.27 H-FABP concentrations below this value are attributed to the inevitable steady release of the protein from muscle cells. In conclusion, we have opened for the first time the possibility of reproducibly producing a recombinant conjugate of a protein antigen with horseradish peroxidase as a marker enzyme for use as a tracer in competitive immunoassays. The applicability of this genetically engineered fusion protein with a defined 1:1 stoichiometry for a clinically relevant analyte, the human heart fattyacid-binding protein, has been shown using plasma from a patient after myocardial infarction. The competitive assay format has two advantages, as compared to the sandwich assay format. First, due to the wide measuring range, the plasma samples in general need not be diluted. Second, the competitive scheme requires only one incubation of samples and tracer in the antibody-coated microtiter plate well instead of the at least two subsequent incubations with samples and detector antibodies in the sandwich-type immunoassays (and, additionally, an enzyme conjugate in indirect assays), thereby reducing the time needed for analysis by 1-2 h. Our approach paves the way for the broad application of the popular peroxidase marker enzyme in competitive immunoassays, employing genetically engineered conjugates. We have already extended the concept by the preparation of a recombinant conjugate of peroxidase and human myoglobin, another analyte important for early detection of myocardial infarction. ACKNOWLEDGMENT This work was supported by the German Federal Ministry of Science and Technology (Grant No. 0310554) and by the European Commission (Grant No. ERB IC15-CT96-1002). A.E. acknowledges funding by Grant No. 4-59 of the Russian State Scientific and Technological Program “Advanced Methods of Bioengineering”. Received for review June 14, 2000. Accepted November 21, 2000. AC000684T Analytical Chemistry, Vol. 73, No. 6, March 15, 2001
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