Langmuir 2005, 21, 6473-6477
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Immunosensing Based on Site-Directed Immobilization of Antibody Fragments and Polymers that Reduce Nonspecific Binding Inger Vikholm-Lundin Information Technology, Technical Research Centre of Finland, P.O. Box 14021, FIN-33101 Tampere, Finland Received December 7, 2004. In Final Form: April 21, 2005 Antibody Fab′-fragments can be directly coupled onto gold, and the space between the fragments can be filled with protein repellent disulfide bearing polymers. Coupling of the antibody Fab′-fragments, and thus both the amount of nonspecific binding and antigen binding but also the ability to regenerate the layer, is dependent on the immobilization procedure. First, the immobilization has taken place by coupling the Fab′-fragments to the surface and thereafter attaching the polymer in the remaining space between the antibodies. Second, the Fab′-fragments have been added after the surface has been coated by polymer. Third, the Fab′-fragments and polymer have been added onto the surface from the same solution. Up to 80% of the antigen could be removed during regeneration, if proper concentrations of polymer and Fab′fragments were immobilized onto the gold surface. Only about 60% of the antigen could be removed, when the fragments were coupled directly onto a clean Au surface before the polymer or if low concentrations of polymer were attached onto gold before the Fab′-fragments. The first immobilization method, however, showed the highest response to antigen.
1. Introduction The use of immunoassay technology in clinical, food safety, and environmental analysis will continue to grow. Traditionally, radioimmunoassay, solid-phase enzyme immunoassay, and fluoroimmunoassay have been used to measure the very specific and tight binding between antibody and antigen. All approaches, however, rely on a marker molecule, such as a radioisotope, an enzyme, or a fluorescent probe, that allows quantification of the antibody-antigen complex. In most cases, the result is not obtained until several incubations, washing, and separation steps have been carried out. Surface plasmon resonance, SPR, on the other hand, does not require a labeled reagent for detection of the molecular interaction. SPR detects changes in refractive index and thickness due to the binding of molecules to a gold surface.1 In immunoassays, antibodies are mostly adsorbed on the sensor surface or covalently coupled via functional groups that are not site-specific. When immobilizing antibodies via amino and carboxyl groups, the orientation of the protein molecule on the sensor surface will be random. The lack of control over the orientation of the antibodies limits the proportion of available binding sites, whereas site-specific immobilization leads to higher activity.2 Options for controlled immobilization of antibodies include specific binding of the Fc region of the antibody to a layer of protein A or protein G.3 Fab′fragments can, moreover, be covalently attached onto supported lipid layers through the free sulfhydryl group opposite the antigen binding domain, and biotinylated antibodies can be coupled onto a surface by biotin/(strept)avidin chemistry.4-9 Assays often suffer of interference * Corresponding author. Tel.: +358 3 3163363. Fax: +358 3 3163319. E-mail:
[email protected]. (1) Jo¨nsson, U.; Fagerstam, L.; Ivarsson, B.; Karlsson, R.; Lund, K.; Lo¨fås, S.; Persson, B.; Roos, H.; Ro¨nnberg, I.; Sjo¨lander, S.; Stenberg, E.; Ståhlberg, C.; Malmqvist, M. BioTechniques 1991, 11, 620. (2) Attili, B.; Suleiman, A. Microchem. J. 1996, 54, 174. (3) Turkova´, J. J. Chromatogr. 1999, 722, 11. (4) Morgan, H.; Taylor, D. M. Biosens. Bioelectron. 1992, 7, 405.
from other molecules that nonspecifically bind to the surface. Tween 20, bovine serum albumin (BSA), casein, fat-free milk, or serum have been used to block nonspecific binding, NSB sites and restrict conformational changes of the immobilized antibodies.10 Yet instead of blocking the surface, it would be preferred to use a host matrix that reduces NSB. The phosphatidyl headgroup is known to suppress NSB.5 Polymers such as poly(ethylene oxide), dextran, and poly(ethylene glycol) have been used to improve the biocompatibility of surfaces and provide an attractive option for producing surfaces that prevent NSB.11-13 To obtain a site-specific orientation of antibodies and prevent nonspecific adsorption, our concept has been to immobilize Fab′-fragments directly onto a gold film and to block the remaining free space by attaching a nonionic hydrophilic polymer of N-[tris(hydroxy-methyl)methyl]acrylamide, pTHMMAA, between the antibodies.14,15 The polymer possesses low NSB and can be covalently attached onto the gold surface by disulfide anchors.16 The immobilization procedure results in a significant reduction (5) Vikholm, I.; Albers, W. M. Langmuir 1998, 14, 3865. (6) Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E. Langmuir 1993, 9, 136. (7) Scho¨nhoff, M.; Lo¨sche, M.; Meyer, M.; Wilhelm, C. Prog. Colloid Polym. Sci. 1992, 89, 243. (8) Krull, U. J.; Brown, R. S.; Vandenberg, E. T.; Heckl, W. M. J. Electron Microsc. Tech. 1991, 8, 212. (9) Heyn, S. P.; Egger, M.; Gaub, H. E. J. Phys. Chem. 1991, 94, 5073. (10) Yang, Z. P.; Li, Y. B.; Balagtas, C.; Slavik, M.; Paul, D. Electroanalysis 1998, 10, 913. (11) Akkoyun, A.; Bilitewski, U. Biosens. Bioelectron. 2002, 17, 655. (12) Pasche, S.; De Paul, S. M.; Voros, J.; Spencer, N. D.; Textor, M. Langmuir 2003, 9, 9216. (13) De Groot, C. J.; Van Luyn, M. J. A.; Van Dijk-Wolthuis, W. N. E.; CadeH, J. A.; Plantinga, J. e A.; Den Otter, W.; Hennink, W. E. Biomaterials 2001, 22, 1197. (14) Vikholm, I.; Sadowski, J. Method and Biosensor for Analysis, Patent Application US2003059954. (15) Vikholm, I. Sens. Actuators, B 2005, 106, 311. (16) Mangeney, C.; Ferrage, F.; Aujard, I.; Marchi-Artzner, V.; Jullien, L.; Olivier Ouari, O.; Djouhar, Re´kaı¨, D.; Laschewsky, A.; Vikholm, I.; Sadowski, J. W. J. Am. Chem. Soc. 2002, 124, 5811.
10.1021/la046992u CCC: $30.25 © 2005 American Chemical Society Published on Web 05/27/2005
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Figure 1. Schematic view of the N-[tris(hydroxy-methyl)methyl]acrylamide, pTHMMAA.
of the NSB of biological macromolecules, because the surface between the Fab′-fragments will be highly hydrophilic, and, moreover, a proper orientation of the antibody fragment will result in a high binding of antigen. The purpose of this study has been to optimize the immobilization procedure by attaching the antibodies and the polymer onto the gold surface in three different ways. First, the antibodies have been attached directly onto the gold surface and the free remaining space has been coated with pTHMMAA. Second, the immobilization order has been the opposite. The polymer has been attached onto the surface before the antibody fragments. Third, the Fab′fragments and the polymer have been attached onto the surface from the same solution. Various concentrations of both Fab′-fragments and polymer have been studied to obtain on optimum amount of antibodies on the sensor surface. 2. Experimental Section Materials. Human IgG (hIgG) and polyclonal goat anti-human F(ab′)2 (from Jackson ImmunoResearch) were used as the model system. The F(ab′)2 fragments were split into Fab′-fragments with dithiotreitol (DTT, Merck) in a 150 mM NaCl, 10 mM HEPES, 5 mM EDTA buffer pH 6.0, typically overnight in a micro-dialysis tube.17 Briefly, F(ab′)2 fragments at a concentration of 0.02-0.5 µg/mL were mixed with HEPES/EDTA buffer and 6.25 mM DTT solution in a micro-dialysis tube. The dialysis tube was immersed in 250 mL of argon-purged HEPES/EDTA buffer and dialyzed overnight at room temperature under argon. The Fab′-fragments were maintained under argon and used immediately. The nonionic hydrophilic polymer, bearing hydroxyl groups, were grafted onto gold by disulfide anchors. The polymer was based on a nonionic monomer, N-[tris(hydroxymethyl)methyl]acrylamide, pTHMMAA (Figure 1). A disulfide-functionalized diazo initiator was employed to incorporate a metal-anchoring moiety in the hydrophilic polymer. pTHMMAA was obtained by free radical homo-polymerization as previously described and had an average molecular weight of 6000 g/mol.16 Measurements. Glass slides coated with a thin film of gold were cleaned in a hot solution of H2O2:NH4OH:H2O (1:1:5) and rinsed with water. The slides were attached via index matching oil to a SPR prism on a Surface Plasmon Resonance Device (SPRDevi, VTT, Tampere, Finland). A flow cell was assembled on the prism, and the flow cell was thoroughly rinsed with 10 mM HEPES, 150 mM NaCl, pH 6.8 buffer solution. The Fab′fragments were allowed to interact with the gold-coated surface typically for 10 min, followed by rinsing the surface with HEPES buffer for 5 min. 1000-1500 µL of pTHMMAA at a concentration of 0.15 g/L was allowed to interact with the surface for 5-10 min. Various concentrations of pTHMMAA in HEPES/EDTA were also grafted onto the gold surface followed by the addition of various concentrations of anti-goat hIgG Fab′-fragments. Alternatively, pTHMMAA was added into the anti-goat hIgG Fab′fragment HEPES/EDTA buffer solution, and both were attached simultaneously onto the gold surface. NSB of the layers was determined with 0.5 g/L bovine serum albumin, BSA. The interaction of the layer with human IgG was studied in a buffer (17) Ishikawa, E. J. Immunoassay 1983, 4, 209.
Figure 2. Change in SPR intensity on attachment of (a) 30 µg/mL goat anti-human IgG Fab′-fragments and (c) 0.15 g/L pTHMMAA onto a thin gold film. Rinsing with buffer is denoted with (b), (d) marks the interaction with BSA, (e) 10 µg/mL hIgG, and (f) regeneration with a glycine-HCl solution. of 50 mM Na2HPO4/NaH2PO4, 150 mM NaCl, pH 7.4. The layers were regenerated with a 0.1 M glycine-HCl solution at pH ) 2.8.
3. Results and Discussion Coupling of Fab′-Fragments Directly onto Au and Blocking of the Remaining Free Surface with pTHMMAA. Goat anti-hIgG Fab′-fragments can be coupled directly onto Au with a high antigen binding efficiency.14,15 In Figure 2, a typical SPR sensor-gram shows the initial fast increase in SPR intensity at a fixed angle on attachment of Fab′-fragments onto gold. The surface was saturated within 10 min, and no Fab′fragments could be rinsed off with buffer. pTHMMAA could be attached on the surface onto which Fab′fragments already had been attached as observed by an additional increase in SPR intensity. Antibody Fab′fragment and polymer immobilization was highly reproducible with a relative standard deviation (RSD) of 2.3% (n ) 3). The increase was, however, dependent on the polymer and Fab′-fragment concentration.15 The polymer was supposed to be attached between the bound Fab′fragments, thus shielding the gold surface from NSB. There was only a minor increase in SPR intensity on adsorption of BSA (0.002 ( 0.001 au). NSB to the pure Fab′-fragment film was 10-fold (0.02 ( 0.005 au). There was a large increase in SPR intensity on interaction of 10 µg/mL hIgG with the layer. This change in SPR intensity corresponded to 0.12 ( 0.02 au. The intensity change was thus higher than that of a film composed of only Fab′fragments, which showed a SPR increase of 0.10 ( 0.005 au when interacting with 100 µg/mL hIgG. Thus, apart from suppressing NSB, the pTHMMAA molecules, moreover, help to increase the response of the layer. The polymer most probably protects the antibody fragments from unfolding, but might also minimize the risk of NSB to the fragment itself. Fab′-fragments that are not covalently bound might also be replaced by polymers. The layer was regenerated with a glycine-HCl solution during 2 min, a washing solution and regeneration time often used to dissociate antigen.18 40% hIgG remained on the surface after regeneration of the Fab′-fragment/ pTHMMAA layer. The disability to regenerate part of the antibodies seems to be closely connected with their orientation. If antibodies are randomly immobilized on (18) Kandimalla, V. B.; Neeta, N. S.; Karanth, N. G.; Thakur, M. S.; Roshini, K. R.; Rani, B. E. A.; Pasha, A.; Karanth, N. G. K. Biosens. Bioelectron. 2004, 20, 903.
Immunosensing Based on Antibody Fragments
Figure 3. Change in SPR intensity on the attachment of 60 µg/mL Fab′-fragments to a surface coated with various amounts of polymer. The inset shows the change in SPR intensity on the attachment of various concentrations of the polymer pTHMMAA onto Au.
the sensor chip, the number of effective sites is reduced and the layer is more difficult to regenerate. Part of the Fab′-fragments was most probably physically adsorbed onto the gold surface, and antigen could for that reason not be removed. Antigen could not be removed from a layer composed of F(ab)2-fragments and pTHMMAA immobilized under the same conditions. The F(ab)2fragments were physically adsorbed and thus randomly oriented on the surface. Biotinylated antibodies that are site-directly immobilized have a functionality of 60%, whereas only 5% of antibodies immobilized by passive adsorption are functional.19 This seems to be the case also for the Fab′-fragment/pTHMMAA layer onto gold. Coupling of pTHMMAA and Subsequent Coupling of Fab′-Fragments. Various concentrations of pTHMMAA were attached onto the gold surface, and antibody Fab′-fragments at a concentration of 60 µg/mL were hereafter immobilized onto the surface. Monolayer formation of the polymer is shown in the inset of Figure 3. Saturation of the surface was obtained at about 0.3 g/L pTHMMAA. The SPR intensity increase corresponds to a monolayer thickness of about 4.4 nm as earlier simulated by fitting SPR data to a Fresnel equation.16 The size of a Fab′-fragment is 7 × 5 × 4 nm3.20 If standing end-on on the surface attached through the thiol group, the Fab′fragments would protrude out from the polymer host matrix, which would enable antigen binding. The amount of Fab′-fragments that attached onto an Au film already coated with pTHMMAA decreased with pTHMMAA concentration as could be expected (Figure 3). If the Au surface was coated with pTHMMAA > 0.015 g/L, no Fab′fragments could be attached. At lower pTHMMAA concentrations, Fab′-fragments attach onto Au probably between the pTHMMAA molecules. Yet as pTHMMAA molecules start to be more closely packed, attachment of Fab′-fragments was hindered. No binding occurred at pTHMMAA concentrations where the polymer starts to form a monolayer. It is known that pTHMMAA gives polymers that are nonionic, very hydrophilic, biocompatible, and, moreover, inert to biological fouling.15 There was no NSB of BSA to the pTHMMAA monolayer. The total change in SPR intensity of layers composed of 60 (19) Davies, J.; Dawkes, A. C.; Haymes, A. G.; Roberts, C. J.; Sunderland, R. F.; Wilkins, M. J.; Davies, M. C.; Tendler, S. J. B.; Jackson, D. E.; Edwards, J. C. J. Immunol. Methods 1994, 167, 263. (20) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753.
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Figure 4. Change in SPR intensity on the attachment of Fab′fragments (9) onto a gold surface coated with various concentrations of pTHMMAA. NSB of 0.5 g/L BSA (3) and interaction with 100 µg/mL hIgG (O). The inset shows the amount of hIgG removed from the pTHMMAA/Fab′-fragment layers after regeneration.
µg/mL Fab′-fragments below a polymer concentration of 0.01 g/L pTHMMAA corresponded to 0.09 ( 0.01 au. Yet the SPR intensity increased to 0.12 ( 0.005 au at 0.3 g/L pTHMMAA. Figure 3 shows that the interaction of hIgG with the monolayer decreased above pTHMMAA concentrations of 0.001 g/L. NSB of BSA decreased with pTHMMAA concentration to zero at concentrations above 0.003 g/L pTHMMAA. Human IgG corresponding to about 0.045 ( 0.002 au could be bound at pTHMMAA concentrations 20 µg/mL (b) Fab′-fragments and pTHMMAA onto gold.
Figure 6. Binding of 100 µg/mL human IgG (O) to a mixed monolayer of Fab′-fragments and pTHMMAA (9).
40% slower than thiols on gold.21 Above a Fab′-fragment concentration of 40 µg/mL, the SPR increase corresponded to 0.14 ( 0.02 au (n ) 9) (Figure 5). When the polymer was attached onto the gold surface before the fragment, the total increase was only 0.09 ( 0.01 au (as shown in the previous section). It therefore seems like the Fab′fragments predominantly were attached on the surface before the polymers. If a large amount of polymers would be attached before the fragments, these would hinder Fab′fragments from reaching the surface and even a low amount of polymers on the surface could be expected to cause a response similar to that of polymers attached on the surface before the fragments as in the previous section. NSB of BSA was dependent on the polymer concentration (Table 1). At a very low polymer concentration (0.001 g/L), a low NSB of BSA could be observed. At 0.006 g/L pTHMMAA, BSA caused a small increase in SPR response when the Fab′-fragment concentration was below 5 µg/ mL or above 70 µg/mL, but between this concentration range there was no NSB of BSA. As the pTHMMAA concentration further increased, it seems like material was removed from the surface. At a polymer concentration of 0.025 g/L, the decrease in response corresponded to 4% of the Fab′/pTHMMAA layer. The decrease in SPR (21) Jung, Ch.; Dannenberger, O.; Yue, X.; Buck, M.; Grunze, M. Langmuir 1998, 14, 1103.
intensity could only be due to a detachment of not covalently bond fragments or polymers from the surface. An exchange with BSA cannot be excluded. Thus, at optimum Fab′-fragment concentration, it seems like NSB to the mixed layer was reduced to -0.004 ( 0.005 au at polymer concentrations in the range of 0.006-0.012 g/L. The NSB binding was remarkably reduced by the polymer as compared to a layer composed of only Fab′-fragments that have a NSB of 0.02 ( 0.005 au, which corresponded to 25% of the specific binding. The response of the mixed Fab′/pTHMMAA monolayer to hIgG was almost independent of Fab′-fragment and polymer concentration in the concentration range studied (Figure 6). The response of hIgG to the layers was 0.08 ( 0.01 au. The response was lower than that of a layer where the antibodies were attached onto the surface before the polymer. A hIgG concentration of 10 µg/mL showed a response of 0.12 ( 0.02 au, but higher than that of a layer where the polymer was attached before the antibody fragments (0.045 ( 0.002 au). Only 34% of the antigen could be removed with a glycine-HCl regeneration solution when the layer was prepared without pTHMMAA from a 5 µg/mL Fab′fragment solution (Figure 7). When the layer was made from higher fragment concentrations, 62 ( 1% of the antigen could be removed, indicating that a higher amount of the fragments were site-directly orientated. A higher amount of fragments seemed to be adsorbed onto the gold surface, and at low Fab′-fragment concentration antibodies might be at least partly unfolded. There was a remarkable increase in the ability to regenerate the layer when polymer was added into the immobilization solution (Figure 7). Even very low pTHMMAA concentrations improved the ability to regenerate the layer. Yet the ability to regenerate the layer was also dependent on the amount of Fab′-fragments in the layer. When 5 µg/mL Fab′fragments were present in the layer, 65 ( 6% of the antigen
Immunosensing Based on Antibody Fragments
could be removed at higher polymers concentrations (Figure 7). 81 ( 1% of the layer could be regenerated when the layer was prepared from low amounts of polymer and 20-40 µg/mL Fab′-fragments. Yet as the pTHMMAA concentration increased, the regeneration efficiency decreased. Above a pTHMMAA concentration of 0.003 g/L, 73 ( 2% of the antigen could be removed from the layer. Too high concentrations of pTHMMAA might hinder Fab′fragments from coupling to the surface through the thiol bond, thereby increasing the amount of fragments adsorbed onto the surface. 4. Conclusions A simple and fast method for immobilizing antibodies onto a gold sensor surface has been optimized for human IgG antibody fragments. The method is generic and can be used for coupling of any antibody in an oriented manner to the sensor surface. Fab′-fragments are coupled directly onto gold, and the remaining space between the fragments is blocked with nonionic, hydrophilic disulfide bearing polymers, pTHMMAA, to suppress nonspecific binding. When the antibody Fab′-fragments were applied onto the
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gold surface and the remaining free space between the antibodies was coated with polymer, a large part of the fragments seemed to be site-directly attached through the free thiol bonds. Part of the fragments was randomly adsorbed onto the surface, and 60% of the antigen could be removed during regeneration of the layer. If pTHMMAA at very low concentrations was first applied on the surface, 60% of the antigen could also be removed. At higher pTHMMAA concentration, the polymer molecules, on the other hand, prevent fragments from attaching onto the surface. When Fab′-fragments and pTHMMAA were immobilized on the surface from the same solution, up to 80% of the antigen could be removed, indicating a high degree of site-directed immobilization of the antibody fragments. The highest response to hIgG was, however, obtained when the antibody fragments were attached onto the surface before the polymer. When the polymer was attached on the surface before the antibody, the response was one-half of that when the antibodies and the polymer were attached from the same solution. LA046992U
