Direct Immobilization of Gold-Binding Antibody ... - ACS Publications

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Direct Immobilization of Gold-Binding Antibody Fragments for Immunosensor Applications Takahisa Ibii,*,† Masaru Kaieda,† Satoru Hatakeyama,† Hidenori Shiotsuka,† Hideki Watanabe,‡,§ Mitsuo Umetsu,‡ Izumi Kumagai,‡ and Takeshi Imamura† Corporate R&D Headquarters, Frontier Research Center, Canon Inc., 30-2, Shimomaruko 3-chome, Ohta-ku, Tokyo 146-8501, Japan, and Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, 6-6-07, Aoba-yama, Aoba-ku, Sendai, Miyagi 980-8579, Japan A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these goldbinding bispecific antibody fragments bind directly to the gold substrate with high affinity (KD ∼ 10-9 M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target proteinbinding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity (KD) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications. Biosensors have been widely applied in various fields such as medical diagnostics, food analysis, environmental monitoring, and as a defense against biohazards.1-5 Immunosensors, a kind of biosensor, are used to detect or quantify disease-related substances known as biomarkers in clinical diagnostics. In immunosensor applications, antibodies are immobilized onto the immunosensor surface to capture specific biomarkers. * To whom correspondence should be addressed. E-mail: ibii.takahisa@ canon.co.jp. Phone: +81-3-3758-2111. Fax: +81-3-3757-3097. † Canon Inc. ‡ Tohoku University. § Present address: National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan. (1) Ionescu, R. E.; Jaffrezic-Renault, N.; Bouffier, L.; Gondran, C.; Cosnier, S.; Pinacho, D. G.; Marco, M. P.; Sanchez-Baeza, F. J.; Healy, T.; Martelet, C. Biosens. Bioelectron. 2007, 23, 549–555. (2) Terry, L. A.; White, S. F.; Tigwell, L. J. J. Agric. Food Chem. 2005, 53, 1309–131. (3) Leonard, P.; Hearty, S.; Brennan, J.; Dunne, L.; Quinn, J.; Chakraborty, T.; O’Kennedy, R. Enzyme Microb. Technol. 2003, 32, 3–13. (4) Paddle, B. M. Biosens. Bioelectron. 1996, 11, 1079–1113. (5) Lim, D. V.; Simpson, J. M.; Kearns, E. A.; Kramer, M. F. Clin. Microbiol. Rev. 2005, 18, 583–607. 10.1021/ac100557k  2010 American Chemical Society Published on Web 04/23/2010

Figure 1. Schematic representation of antibody fragment immobilization by different methods. Scheme a shows conventional immobilization with physical adsorption (a-1) and chemical crosslinking (a-2). This immobilization scheme is widely used; however, the orientation of the immobilized antibody fragments is heterogeneous. Scheme b shows immobilization with bispecific antibody fragments that are themselves immobilized via gold-binding antibody fragments.

There is an increasing need for today’s immunosensors to efficiently immobilize various antibodies at high density so that they demonstrate improved sensitivity and a reduction in size. To meet this requirement, several groups have reported that smaller recombinant antibody fragments, which consist of only the antigen binding domain of the antibody, are useful as capture probes for immunosensors.6,7 Antibody fragments, including the variable fragment (Fv), which consists of a heavy chain variable region (VH) and a light chain variable region (VL), and each antibody domain, VH or VL, have been considered as smaller alternative molecules with an equivalent binding affinity and binding capability for target molecules as their parent antibodies. A critical consideration is the development of a technique to immobilize antibody fragments on the sensor substrate surface without decreasing their binding affinities and binding capacities. The immobilization of antibody fragments is conventionally achieved using physical adsorption or covalent cross-linking methods, as shown in Figure 1a. In the physical adsorption method, antibody fragments are immobilized through mainly hydrophobic and hydrophilic interaction with the sensor substrate at random. In this method, the orientation of the antibody (6) Howell, S.; Kenmore, M.; Kirkland, M.; Badley, R. A. J. Mol. Recognit. 1998, 11, 200–203. (7) Steinhauer, C.; Wingren, C.; Khan, F.; He, M.; Taussig, M.; Borrebaeck, C. A. K. Proteomics 2006, 6, 4227–4234.

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fragments on the sensor substrate surface cannot be controlled, and therefore, the antibody fragments lose their binding activity to the target molecules. In the covalent cross-linking method, the free-amino groups on the antibody can be randomly coupled with several reactive moieties on the sensor substrate surface. However, as a result of the random coupling, the orientation of the immobilized antibody fragments is also random. Thus, there is an increasing need for a technique of achieving well-oriented immobilization of antibody fragments in a small area without decreasing their binding affinity for target molecules, especially for miniaturized diagnostic devices. We have developed a method to directly immobilize antibody fragments on a sensor substrate using molecular recognition. We fabricated recombinant bispecific antibodies8 consisting of two kinds of antibody fragments, one for the sensor substrate material and the other for a target molecule. The bispecific antibody fragments were bound directly to the sensor substrate via their substrate-binding domain. Therefore, well-oriented immobilization of the bispecific antibody fragments on the sensor substrate was expected (Figure 1b). In this study, to achieve proof of concept, we fabricated bispecific antibody fragments that combined an antigold antibody fragment9 with an antibody fragment specific to a target protein. Gold is widely used as an immunosensor material in several applications, including as surface plasmon resonance (SPR) sensor substrates,10 in electrodes for electronic measurements,11-13 and as a protein marker label such as colloidal gold nanoparticles.14-16 In this regard, it is expected that gold-binding bispecific antibody fragments would be applicable to a wide variety of immunosensors. SPR analyses showed that the bispecific antibody fragments were able to bind directly to the gold substrate with high affinity and subsequently bind to target proteins. The affinity distribution of the immobilized bispecific antibody fragments was assessed using the Sips isotherm.17 These results indicate that the bispecific antibody fragments were able to achieve homogeneous immobilization and maintain their target protein-binding capability. EXPERIMENTAL SECTION Reagents. Hen egg lysozyme (HEL) was purchased from Seikagakukogyo Ltd. (Tokyo, Japan). Human prostate-specific antigen (PSA) was purchased from IPAC Ltd. (Sittingbourne, Kent, England). Recombinant human chorionic gonadotropin (hCG) was purchased from Acris Antibodies (Hiddenhausen, Germany). Recombinant tumor necrosis factor-R (TNF-R) was purchased from Strathmann Biotech (Hamburg, Germany). Guanidine hydrochloride (GdnHCl), L-arginine, and glutathione disulfide (GSSG) (8) Holliger, P.; Prospero, T.; Winter, G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6444–6448. (9) Watanabe, H.; Nakanishi, T.; Umetsu, M.; Kumagai, I. J. Biol. Chem. 2008, 283, 36031–36038. (10) Timothy, T. E.; Lewis, J. N. Langmuir 1995, 11, 4177–4179. (11) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977–989. (12) Creager, S. E.; Olsen, K. G. Anal. Chim. Acta 1995, 307, 277–289. (13) Zhou, L.; Ou, L. J.; Chu, X.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2007, 79, 7492–7500. (14) Endo, T.; Kerman, K.; Nagatani, N.; Hiepa, H. M.; Kim, D. K.; Yonezawa, Y.; Nakano, K.; Tamiya, E. Anal. Chem. 2006, 78, 6465–75. (15) Maier, I.; Morgan, M. R.; Lindner, W.; Pittner, F. Anal. Chem. 2008, 80, 2694–2703. (16) Zhao, W.; Brook, M. A.; Li, Y. ChemBioChem 2008, 9, 2363–2371. (17) Sips, R. J. J. Chem. Phys. 1948, 16, 490–495.

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Figure 2. Expression vector and plasmid vectors used to produce the bispecific antibody framgents, monospecific antibody fragments for conventional immobilization and gold-binding peptide (GBP)-fused antibody fragments.

were purchased from Wakojyunyaku Kogyo Co., Ltd. (Osaka, Japan). The amine coupling kit (containing 0.1 M NHS, 0.4 M EDC, and 1 M ethanolamine (pH 8.5)) and bare and carboxymethyl dextran-coated gold sensor substrates for SPR measurement were supplied by GE Healthcare UK Ltd. (Little Chalfont, Buckinghamshire, England). Plasmid Construction. The pRA2 expression vector used in this study is shown in Figure 2a. The pRA218 expression vector includes the T7-promoter, a bacterial neo fusion gene, the ShineDalgarno sequence, the pelB leader sequence, and the six repeated histidine, (His)6-tag sequence for protein purification. The bispecific antibody fragments used in this study were constructed from a gold-binding antibody fragment which was connected to a target protein-binding antibody fragment using a peptide linker of 28 amino acids (Figure 2b). The gold-binding antibody fragment was derived from an antibody heavy chain variable region (VH) of the human anti-gold antibody.9 The following were employed as target binding antibody fragments: a scFv (single-chain variable fragment19) specific to hen egg lysozyme (HEL)20 and variable heavy chain antibodies (VHHs) (camelid-derived recombinant antibody fragments) comprising a single domain, the so-called VH domain of the camelid heavy chain antibody21 specific to a disease-related protein marker selected from a prostate specific antigen (PSA),22 human chorionic gonadotropin (hCG),23 and tumor necrosis factor-R (TNF-R).24 By way (18) Makabe, I.; Asano, R.; Ito, T.; Tsumoto, K.; Kudo, T.; Kumagai, I. Biochem. Biophys. Res. Commun. 2005, 328, 98–105. (19) Plu ¨ ckthun, A. Immunol. Rev. 1992, 130, 151–188. (20) Silverton, E. W.; Padlan, E. A.; Davies, D. R.; Smith-Gill, S.; Potter, M. J. Mol. Biol. 1984, 180, 761–765. (21) Muyldermans, S. J. Biotechnol. 2001, 74, 277–302.

of comparison with the immobilization by bispecific antibody fragments, genes encoding target protein-binding monospecific antibody fragments for conventional immobilization (Figure 2c) and the multiple tandem repeated gold-binding peptide (GBP, MHGKTQATSGTIQS, 14 amino acid sequence)25 fused to the C-terminus of the recombinant antibody fragments (Figure 2d) were also constructed and inserted into the pRA2 vector. Production and Purification. The production and purification schemes for each bispecific antibody fragment are shown in Supplemental Figure 1 in the Supporting Information. The E. coli strain BL21 (DE3) transformed with the expression vector encoding bispecific antibody fragments was precultured at 28 °C in 3 mL of 2× YT medium containing 100 µg/mL ampicillin. The precultured E. coli cells were subcultured to a 500 mL bacteria culture flask containing fresh 250 mL 2× YT medium supplemented with 100 µg/mL ampicillin, and then the culture was incubated at 28 °C with shaking at 140 rpm. When the optical density (OD) of wavelength 600 nm exceeded 0.8, isopropyl-β-Dthiogalactopyranoside (IPTG) to a final concentration of 1 mM was added to induce the expression of the antibody and the culture was incubated overnight at 28 °C while shaking at 140 rpm. Harvested cells were collected by centrifuging at 6000 rpm for 30 min. The collected cells were suspended in 20 mL of 20 mM Tris-HCl buffer (pH 8.0) with 500 mM NaCl and then homogenated by sonication for 30 min on ice. After centrifugation at 13 000 rpm for 30 min, the collected inclusion bodies were denatured with 20 mM Tris-HCl (pH 8.0) buffer containing 6 M guanidine hydrochloride (GdnHCl), and the suspension was centrifuged at 13 000 rpm for 30 min at 4 °C. The denatured bispecific antibody fragments in the supernatant were prepurified by using His-Bind resin (Novagen, Merck Chemical Ltd., Nottingham, Notts, U.K.) following the manufacturer’s protocol. The refolding of the denatured bispecific antibody fragments was carried out by a stepwise dialysis method reported by Umetsu et al.26 Briefly, the concentration of the unfolded bispecific antibody fragments was adjusted to 7.5 µM in the 50 mM Tris-HCl (pH 8.0) buffer with 6 M GdnHCl, 200 mM NaCl, and 1 mM EDTA, and the bispecific antibody fragments were reduced by the addition of 2-mercaptoethanol (2-ME) at a 50-fold molar excess relative to the protein. After 2-ME was removed by dialysis against the same Tris-HCl buffer without 2-ME, the unfolded bispecific antibody fragments were refolded by gradual removal of GdnHCl by means of stepwise dialysis from 6 to 0 M through 3 M, 2 M, 1 M, and 0.5M. GSSG (375 µM) and 0.4 M L-arginine were added at the 1 and 0.5 M GdnHCl stages, respectively. After the refolding step, gel filtration chromatography (AKTA 10S, Superdex 200 10/ 300GL) was carried out in 50 mM Tris-HCl buffer containing 200 mM NaCl to purify the monomeric form of the bispecific antibody fragments. Sodium dodecyl sulfate-polyacrylamide gel electro(22) Saerens, D.; Kinne, J.; Bosmans, E.; Wernery, U.; Muyldermans, S.; Conrath, K. J. Biol. Chem. 2004, 279, 51965–51972. (23) Van der Linden, R. H.; Frenken, L. G.; de Geus, B.; Harmsen, M. M.; Ruuls, R. C.; Stok, W.; de Ron, L.; Wilson, S.; Davis, P.; Verrips, C. T. Biochim. Biophys. Acta 1999, 1431, 37–46. (24) Coppieters, K.; Dreier, T.; Silence, K.; de Haard, H.; Lauwereys, M.; Casteels, P.; Beirnaert, E.; Jonckheere, H.; Van de Wiele, C.; Staelens, L.; Hostens, J.; Revets, H.; Remaut, E.; Elewaut, D.; Rottiers, P. Arthritis Rheum. 2006, 54, 1856–1866. (25) Brown, S. Nat. Biotechnol. 1997, 15, 269–272. (26) Umetsu, M.; Tsumoto, K.; Hara, M.; Ashish, K.; Goda, S.; Adschiri, T.; Kumagai, I. J. Biol. Chem. 2003, 278, 8979–8987.

phoresis (SDS-PAGE) and Western blotting were carried out to confirm the purity of the bispecific antibody fragments. Antibody fragments other than bispecific antibody fragments as shown in parts c and d of Figure 2 were prepared from culture supernatant.27 Briefly, the E. coli strain BL21 (DE3) transformed with the expression vector encoding the antibody fragments was cultured as described above. The cultured medium was centrifuged at 6000 rpm for 30 min. Ammonium sulfate was slowly added to the resultant supernatant to 50% saturation at 4 °C. The precipitate was suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and the Protease Inhibitor Cocktail (Nacalai Tesque, Inc., Kyoto Japan). The suspension was centrifuged at 12 000 rpm for 30 min, and the corrected clear supernatant was dialyzed against Tris-HCl buffer (pH 8.0) containing 500 mM NaCl. The dialyzed supernatant was loaded onto His-Bind Resin, and then the prepurified antibody fragments were purified by GFC as shown above. Binding Analysis. All binding analyses in this study were conducted using a SPR biosensor (BIAcore X, GE Healthcare). Before assessment of the binding ability, the nontreated gold sensor substrates (SIA-kit Au, GE Healthcare) were first incubated with concentrated hydrochloric acid overnight, then ultrasonicated in acetone and iso-propanol for 15 min, and finally rinsed with copious amounts of deionized water. All of the binding analyses using the SPR biosensor were carried out in PBS+ 0.1 wt % Tween 20 (0.1% PBS-T) at 25 °C at a flow rate of 20 µL/min. After the cleaned gold sensor substrates were equilibrated with 0.1% PBS-T, 40 µL of bispecific antbody fragment solution at various concentrations was immobilized on the gold sensor substrate. The sensor substrate was then washed with buffer to remove the excess bispecific antibody fragments, and then 40 µL of target protein (TNF-R, PSA, hCG, HEL) at various concentrations was loaded onto the sensor substrate under the same buffer conditions. To calculate the kinetic parameters, curve fitting was conducted using BIAevaluation software (GE Healthcare). Comparison of Target Protein-Binding Efficiencies. To compare the target protein-binding efficiency, monospecific antibody fragments were immobilized on the gold sensor substrate by two different methods: (1) physical adsorption and (2) covalent cross-linking. For physical adsorption, monospecific antibody fragment solution (5 µM) in PBS (without Tween 20) was loaded onto the cleaned gold sensor substrate at a flow rate of 5 µL/ min. After immobilization, the sensor substrate was washed with 0.1% PBS-T at 20 µL/mL until the SPR signal became stable. Covalent cross-linking was performed by amine-coupling on a dextran-coated sensor substrate, CM5, in accordance with the manufacturer’s instructions. In these two immobilization methods, the number of immobilized monospecific antibody fragments on the sensor substrate was controlled at the same level as that of the bispecific antibody fragments. The binding efficiency, which is the ratio of the maximum number of antibody fragments bound to the target proteins of all antibody fragments on the gold substrate (eq 1), was calculated by the method of Renberg et al.28 with a slight modification. (27) Skerra, A.; Plu ¨ ckthun, A. Science 1988, 240, 1038–1041. (28) Renberg, B.; Shiroyama, I.; Engfeldt, T.; Nygren, P. K.; Karlstrom, A. E. Anal. Biochem. 2005, 341, 334–343.

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target protein efficiency (%) )

X(RU) Mw(antibody) × 100 Y(RU) Mw(target protein) (1)

Here, X is the calculated maximum SPR response of the target protein (Rmax), Y is the SPR response of the immobilized capture molecule (RU) shown in Supplemental Figure 2 in the Supporting Information, Mw(antibody) is the molecular weight of the immobilized antibody fragment, and Mw(target protein) is the molecular weight of the target protein. Homogeneity Analysis. The Sips isotherm29 (eq 2) was used to assess the heterogeneity of affinity (KD) of antibody fragments immobilized on the gold substrate surface. f)

(c/KD)a 1 + (c/KD)a

(2)

Here, f is the functional coverage of binding sites of each antibody fragment on the gold substrate surface at equilibrium, c is the concentration of unbound target protein, and a is the index related to the distribution of KD of each antibody fragment on the gold substrate surface with respect to the target protein. If the a-value is 1, the affinity of the antibody fragment is homogeneous. A smaller a-value indicates a wider distribution of KD, which tends to increase heterogeneity. Using kinetic data obtained from SPR analysis, we compared the resulting a-values to evaluate the heterogeneity of the immobilized bispecific antibody fragments and monospecific antibody fragments by physical adsorption or covalent cross-linking.

Figure 3. (a) SDS-PAGE (12.5% polyacrylamide) and Western blotting analysis of TNF-R-binding bispecific antibody fragments with or without a reducing agent (2-mercaptoethanol, 2-ME). After SDSPAGE, the bispecific antibody fragments transferred with a PVDF membrane were probed with the horseradish peroxidase-conjugated anti-C-term (His)6 antibody and then detected by chemiluminescence. Lane 0, nickel affinity column-purified bispecific antibody fragments; lane M, protein size markers; and lanes 1-12, eluted fractions after gel-filtration chromatography, are shown in part b. Solid arrows show the monomer form of the bispecific antibody fragments.

RESULTS AND DISCUSSION Production and Purification of Bispecific Antibody Fragments. Bispecific antibody fragments consisting of the goldbinding VH9 and target protein-binding antibody fragments were produced by an E. coli expression system. Bispecific antibody fragments were obtained by refolding and purifying inclusion bodies that are insoluble and inactive protein aggregates containing highly enriched unfolded recombinant proteins. After solubilization of the inclusion bodies with denaturant, the bispecific antibody fragments were purified using nickel affinity column chromatography. Then, the purified bispecific antibody fragments were refolded by gradually removing the denaturing reagent GdnHCl using stepwise dialysis to obtain soluble and biologically active bispecific antibody fragments. Figure 3 shows the results of gold and TNF-R-binding bispecific antibody fragments. SDSPAGE and Western blotting showed that a large amount of the nickel affinity column-purified bispecific antibody fragments formed soluble aggregates through inappropriate intermolecular disulfide bonds (Figure 3a (lane 0)). Gel filtration chromatography (GFC) was conducted to separate the soluble aggregates from the monomer form of the bispecific antibody fragments. As shown in Supplemental Figure 3 in the Supporting Information, two monomer forms were isolated in the HEL-binding bispecific antibody fragment. This result indicated that a non-native isoform was obtained due to inappropriate intramolecular disulfide bonds. In contrast, such an

isoform was not detected in Figure 3a. This suggests that VHH forms might be suitable for the target protein-binding domain of the gold-binding bispecific antibody fragments because VHHs are more stable and have simpler folding properties.30 Because the bispecific antibody fragments from monomer fractions 9-11 showed superior binding capacity to the gold substrate than that those obtained from fractions 5-8 (data not shown), these fractions were used for subsequent experiments. The final yield of the purified bispecific antibody fragments was estimated to be about 1 mg/L culture medium, as determined by measuring the UV absorption at 280 nm. Binding Capability of Bispecific Antibody Fragments to a Gold Substrate. SPR analysis was conducted to evaluate the binding capability of the bispecific antibody fragments. The scheme of the SPR analysis is shown in Supplemental Figure 2 in the Supporting Information. Bispecific antibody fragments were found to directly immobilize onto the gold substrate and subsequently bind their target proteins as shown in Figure 4a. The SPR signal response increased in proportion to the concentration of bispecific antibody fragments. By contrast, only a slight increase in the signal for the monospecific antibody fragment that did not contain the gold-binding domain was observed in Figure 4b (blue line). The monospecific antibody fragment as shown in Figure

(29) Vijayendran, R. A.; Deborah, E.; Leckband, D. E. Anal. Chem. 2001, 73, 471–480.

(30) Arbabi-Ghahroudi, M.; Desmyter, A.; Wyns, L.; Hamers, R.; Muyldermans, S. FEBS Lett. 1997, 414, 521–526.

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Figure 5. Target protein-binding efficiency of immobilized bispecific antibody fragments (red) and monospecific antibody fragments immobilized by physical adsorption (blue) and covalent cross-linking (yellow).

Figure 4. (a) SPR sensorgram showing the immobilization of bispecific antibody fragments on the gold substrate and subsequent capture responses of the target protein (TNF-R) and the nontarget protein (BSA). (b) SPR sensorgram of different concentrations of bispecific antibody fragments binding to the gold substrate and antibody fragment for TNF-R. (c) SPR sensorgram of different concentrations of target protein (TNF-R) binding to immobilized bispecific antibody fragments.

2c, had a (His)6 sequence at the C-terminus. Previously, Peelle et al. reported that yeast expressing (His)6 on the cell surface was able to bind to gold.31 However, in this experiment, the binding of antibody fragments to the gold substrate via the Histag was suppressed, probably due to the lack of an avidity effect. These results indicate that the bispecific antibody fragments were bound specifically to the gold substrate via their gold-binding domain. The affinity constant (dissociation constant, KD) of the bispecific antibody fragments with respect to the gold substrate was calculated to be in the order of the nanomolar (2.5 × 10-9 M) range, which was slightly decreased compared with that for the gold-binding VH. This indicates that the gold-binding capability of the gold-binding VH was maintained in the bispecific antibody fragment format. Target Protein-Binding Efficiency of Immobilized Bispecific Antibody Fragments. After immobilization of the bispecific antibody fragments onto the gold sensor substrate, continuous SPR analysis was conducted to evaluate the target protein-binding efficiency of the immobilized bispecific antibody fragments (Supplemental Figure 2 (Schemes 3-5) in the Supporting Infor(31) Peelle, B.; Krauland, E.; Wittrup, D.; Belcher, A. M. Langmuir 2005, 21, 6929–6933.

mation). As shown in Figure 4a and 4c, the SPR signal increased following injection of the target protein. However, when a bovine serum albumin (BSA) solution was injected as a nontarget substance, the signal increased slightly. The affinity constant of the immobilized bispecific antibody fragment to the target protein (KD, 3.8 × 10-9 M) was of the same order as the bispecific antibody fragment in the free nonimmobilized state (KD, 4.3 × 10-9 M). Thus, the bispecific antibody fragments maintained their specific target protein-binding capability even when immobilized on the gold substrate. The binding efficiency was estimated from eq 1.28 Here, four kinds of bispecific antibody fragments were produced, in which each bispecific antibody fragment had a different target protein-binding domain. The results are shown in Figure 5. Approximately 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target proteinbinding efficiency, regardless of the target proteins. The target protein-binding efficiency of the bispecific antibody fragments was compared with conventional immobilization methods. The binding efficiency using these conventional methods was generally lower than that of the bispecific antibody fragments (Figure 5). Relatively high values were observed for the covalently immobilized monospecific anti-TNF-R antibody fragments. The reason for this is unclear, but the immobilized conditions might be especially suitable for the anti-TNF-R antibody fragments. Except for the covalently immobilized monospecific anti-TNF-R antibody fragments, the bispecific antibody fragments were found to have about a 3-fold higher binding efficiency than that for the conventionally immobilized monospecific antibody fragments. Antibody fragments directly immobilized onto solid phase materials generally show a reduced binding ability,32 which may be due to their random orientation and/or denaturation caused by the interaction between the sensor substrate materials and the immobilized antibody fragments. We believe that the high target protein-binding efficiency of the bispecific antibody fragments resulted from oriented immobilization. However, there was no significant affinity reduction of the monospecific antibody fragments that were immobilized on the gold substrate by conventional methods. The KD of immobilized bispecific antibody fragments against TNF-R was (32) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Anal. Biochem. 2003, 312, 113–124.

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Figure 6. Assessment of the distribution in KD was carried out using the Sips isotherm (eq 2). (a)Immobilized bispecific antibody fragments (KD, 3.8 × 10-9). (b) Monospecific antibody fragments immobilized by covalent cross-linking (KD, 4.7 × 10-9). (c) Monospecific antibody fragments immobilized by physical adsorption (KD, 4.7 × 10-9). Red squares show analysis points of each immobilization. Solid lines show the Sips isotherm of each “a”-value in evaluated KD.

3.8 × 10-9 and that of TNF-R-binding monospecific antibody fragments for physical and chemical adsorption was 4.7 × 10-9. So, we attempted to evaluate the affinity distribution of the immobilized bispecific antibody fragments to further evaluate their characteristics. Using kinetic data obtained from the SPR analysis, we applied the Sips isotherm29 (eq 2) to assess the homogeneity of affinity (KD) for each antibody fragment on the gold substrate. The results of the Sips analysis of the bispecific antibody fragments bound to TNF-R, compared with those for the TNF-Rbinding monospecific antibody fragments immobilized by physical or covalent cross-linking, are shown in Figure 6a-c. The evaluated index “a”, as determined by Sips analysis for the bispecific antibody fragments immobilized on gold, was approximately 1 (Figure 6a), which indicates that homogeneous immobilization was achieved with bispecific antibody fragments. By contrast, the evaluated index “a” for monospecific antibody fragments immobilized by covalent cross-linking was lower (“a” is about 0.28), as shown in Figure 6b. The results of the monospecific antibody fragments immobilized by physical adsorption, as shown in Figure 6c, were a poor fit to the Sips isotherm because the binding efficiency was much lower than that estimated from the affinity constant. These values were much lower than previously reported.29 The reason for this is unknown, but it may be because of the use of monovalent antibody fragments. The Sips isotherm 4234

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assumes that binding site heterogeneity is a Gaussian distribution. The poor fit to the Sips model might be because of the broad heterogeneous distribution of affinity obtained using this conventional immobilization method. Temirov et al. analyzed the affinity distribution of covalently immobilized full-length antibodies using the single molecule approach.33 They reported that the covalently immobilized antibodies, especially monovalent antibodies, represent a broad affinity distribution due to heterogeneity and steric hindrance. Several groups have recently reported that some proteins could be immobilized on substrates using substrate material-recognition peptides.25,34-39 Of note, the gold-binding peptide (GBP), which has been selected using cell surface display screening,25 has been investigated to immobilize proteins for biosensor applications.40-42 To evaluate alternative immobilization methods, other than the method using the gold-binding antibody fragment by molecular recognition, we prepared GBP-fused antibody fragments comprising GBP, 3R-GBP (three tandem repeats of GBP), and 7R-GBP, which were connected to a target protein-binding antibody fragment as a fusion partner (Figure 2d). The KD of the GBP, 3R-GBP, and 7R-GBP connected antibody fragments to the gold substrate were 9.8 × 10-7, 3.7 × 10-8, and 1.3 × 10-8 M, respectively, which were at a comparable level to that of the previous report by Tamerler et al.40 The KD values were about one-order higher (weaker affinity) than that of the bispecific antibody fragments. This higher affinity for the gold sensor substrate is useful for practical immunosensor applications because it can reduce not only the amount of capture molecules but also the immobilization time. Subsequent evaluation of the target protein-binding efficiency of the immobilized GBP fusion antibody fragments was about 40%, which is 2 times lower than that of the bispecific antibody fragments. Moreover, the Sips isotherm showed that the GBP-fused antibody fragments revealed a more heterogeneous affinity distribution. Recently, Kacar et al. reported that multiple tandem-repeats (n ) 5, 6, 7, 9) of GBP fused to the N-terminus of alkaline phosphatase (AP) was immobilized on the gold substrate and the AP activity evaluated. In their study, the 5RGBP-fused AP was found to be the best gold-binding protein, possessing the best AP activity among the multiple GBP-fused AP.41 Therefore, there is room for further investigation of the differentiation of gold-binding capability and target protein-binding capability between bispecific antibody fragments and GBP-fused antibody fragments. In addition, these (33) Temirov, J. P.; Bradbury, A. R.; Werner, J. H. Anal. Chem. 2008, 80, 8642– 8648. (34) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665–668. (35) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169–172. (36) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577–585. (37) Thai, C. K.; Dai, H. X.; Sastry, M. S. R.; Sarikaya, M.; Schwartz, D. T.; Baneyx, F. Biotechnol. Bioeng. 2004, 87, 129–137. (38) Sano, K.; Sasaki, H.; Shiba, K. J. Am. Chem. Soc. 2006, 128, 1717–1722. (39) Gaskin, D. J. H.; Starck, K.; Vulfson, E. N. Biotechnol. Lett. 2000, 22, 1211– 1216. (40) Tamerler, C.; Oren, E. E.; Duman, M.; Venkatasubramanian, E.; Sarikaya, M. Langmuir 2006, 29, 7712–7718. (41) Kacar, T.; Zin, M. T.; So, C.; Wilson, B.; Ma, H.; Gul-Karaguler, N.; Jen, A. K.; Sarikaya, M.; Tamerler, C. Biotechnol. Bioeng. 2009, 103, 696–705. (42) Verde, A. V.; Acres, J. M.; Maranas, J. K. Biomacromolecules 2009, 10, 2118–2128.

results might be because of a differentiation in the structure of the gold-binding domains. The gold-binding domain of the bispecific antibody fragment consists of about 120 amino acids and has a relatively rigid β-sandwich structure. By contrast, GBP, which comprises 14 amino acids, is thought to have a relatively smaller and more flexible structure.42 In this respect, we consider that the rigid structure of the antibody format might work as a spacer to maintain a certain distance between the antibody fragment and the gold substrate, thereby suppressing the denaturation of the bispecific antibody fragment target protein-binding domain. CONCLUSIONS We have developed a novel immobilization platform for immunosensor applications that features genetically engineered bispecific antibody fragments. SPR analyses showed that these bispecific antibody fragments were directly immobilized onto the gold substrate with high affinity, retaining about a 3-fold higher binding efficiency than that achieved using other conventional methods. The target protein binding of the immobilized bispecific antibody fragments was in good agreement with the Sips isotherm. These results demonstrate that bispecific antibody fragments can (43) Schnirman, A. A.; Zahavi, E.; Yeger, H.; Rosenfeld, R. Nano Lett. 2006, 6, 1870–1874. (44) Watanabe, H.; Tsumoto, K.; Taguchi, S.; Yamashita, K.; Doi, Y.; Nishiyama, Y.; Kondo, H.; Umetsu, M.; Kumagai, I. Bioconjugate Chem. 2007, 18, 645– 651. (45) Hattori, T.; Umetsu, M.; Nakanishi, T.; Tsumoto, K.; Ohara, S.; Abe, H.; Naito, M.; Asano, R.; Adschiri, T.; Kumagai, I. Biochem. Biophys. Res. Commun. 2008, 365, 751–757.

provide an immobilization method that retains the binding ability and homogeneity of the binding affinity. In addition, GBP was evaluated as an alternative immobilization method using molecular recognition. Target protein-binding efficiency of immobilized GBP-fused antibody fragments was 2 times lower than that of the bispecific antibody fragments, and their affinity distribution revealed increased heterogeneity. Immobilization of bispecific antibody fragments on a substrate sensor may offer a potential platform because of their highperformance immobilization and simple immobilization procedure without sensor modification. Recently, antibody fragments that recognize nonbiological substances such as metals and polymers have been obtained using in vitro screening techniques.43-45 Therefore, the bispecific antibody fragment immobilization system described here could be applied to various sensor materials other than gold. We believe that this bispecific antibody fragment system is especially suitable for immunosensor applications such as in antibody microarrays and nanocolloidal sensors, in which it is important to immobilize the active antibody fragment at a high density in a very small area. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 1, 2010. Accepted April 13, 2010. AC100557K

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