Langmuir 1993,9, 136-140
136
Antigen Binding to a Pattern of Lipid-Anchored Antibody Binding Sites Measured by Surface Plasmon Microscopy B. Fischer,+S. P. Hem,* M. Egger,, and H. E. Gaub’ Physik Department der Techniechen Universitat Miinchen, 0-8046 Garching, Germany Received February 28,1992. I n Final Form: July 8,1992 Fab’ fragmenta of a monoclonal antibody against a dinitrophenolhapten (DNP hapten) were covalently bound to a phospholipid. A monomolecular film of a mixture of this Fab-lipid with a pure phospholipid (DMPC) was allowed to form at the air-water interface starting from a vesicle suspension. Thie film was separated from the vesicle phase and driven into a two-dimensionalphase separation by lateral compreasion. After large domains, rich in Fab-lipids, had formed, the monolayer was transferred onto a high refractive index glass prism which was precoated with a thin gold layer. The domain formation as well as their transfer was controlled by microfluorimetry. The domain pattern waa shown to be preserved after the transfer procedure. Imaging by surface plasmon microscopy (SPM) confirmed that the films had monomolecular thickness. The measured height step between protein-richdomains and the surrounding phospholipid matrix agreed well with the data known for the dimensions of crystallized Fab’ fragmenta. DNP-labeled bovine serum albumin (DW-BSA) was used aa an antigen and was allowed to bind to the supported membranes. The increase in the thickness of the film upon antigen binding was measured and compared with the nonspecific binding of BSA. Subsequent incubation with an anti-DNP antibody lead to an additionalincrease in the domain thickness aa did the further incubation with an antimouseantibody. By meane of this work we showed that phase-segregated lipid-protein membranes can be designed in such a way that they form a highly specific two-dimensional recognition pattern on solid surfaces. SPM ie an extremely sensitive technique which, in combination with a recognition pattern, may ale0 be applicable for sensor devices. may be chosen such that nonspecific adsorption is minimized, (ii) receptors may be incorporated into the membrane in a well-defined density and orientation, and (iii) the lateral distribution of the components may be controlled, such that a recognition pattern can be designed on the sensor surface. In general, the information, which one wants to derive from the output of a sensing device, is the number of specifically bound molecules of interest relative to the number of the nonspecifically bound molecules. Ordinarily both quantities must be derived in different experiments in order to determine the so-called binding contrast. The use of a recognition pattern has the advantagethat the binding contrast is directly measurable on one sample, provided the sensing device allow spatial or lateral resolution. Here we have employed surface plasmon microscopy (SPM) and surface plasmon spectroscopy (SPS),techniques which have previously been used in biosensor applications4and for monitoring streptavidin bindingesWe built a device which allow the imaging of a thin metal surface with a lateral resolution of a few micrometers at an accuracy normal to the metal film of some angstroms.e The potential uses of this technique are not fully exploited yet. In this paper we make use of the change in the optical thickness of the dielectric layer in contact with a gold surface upon binding of the ligands.
Introduction Mondodantibodieaare clearlythe molecules of choice for moet molecular recognition purposes. However, most of the highly sensitive sensor devices are surface sensitive which requires that the antibodies be immobilized on solid supports. Many techniques have been developed in which a thin film of the receptor protein is formed on the surface of the device (for a review see ref 1). We have introduced a new approach in which the binding site of a monoclonal antibody was fused to the head group of a phospholipid, resulting in a semisynthetic receptor with custom-designable bindingcharacteristics.2 Langmuh-Blodgett (LB) layers containing such receptors3seem ideally suited films for surface-sensitive optical devices since for such devices a film of monomolecular thickness and well-defined orientation of the receptors is desirable. Moat sensor surfaces are also subject to nonspecific adsorption. This gives rise to a decreased sensitivity as well as a diminished specificity of the device. When designingsensor surfaces of high specificityand sensitivity, one should recall that proteins and polypeptides, which are the moet interesting molecules to be detected in many applications, have evolved together with the cell membranes and therefore only weakly interact nonspecifically with natural lipids. It therefore seems to be a sensible approach to use reconstituted cell membranes or similar systems as the interface between the solid surface of the sensing device and the sample lumen. This approach has three obvious advantages: (i) the lipid of this membrane
Material and Methods Fab-Lipid. A detailed description of the synthesis of the Fab-lipid (Figure 1) is given in ref 2. Briefly monoclonal antibodies’ of subtype IgGl were derived from the cell line AN02 and labeled, following standard procedures, with an average of four molecules of Texas Red. (Fab’lz fragmente were prepared
To whom correspondence should be addreseed. + Present address: Max Planck Institut fijr Polymerforschung,
D-6500Mainz, Germany. t Present address: Boehringer Mannheim Gmbh, D-8132Tutzing, Germany. f Present address: VDI/VDE Technologiezentrum, D-1000Berlin 30,Germany. (1)Scouten, W. H. In Method8 in Enzymology; K. Mosbach, Ed.;
(4) Kooyman, R. P. H.; Kolkman,H.; Gent, J.; Greve, J. Anal. Chem. Acta 1988,213, 35-45. ( 5 ) HBussling, L.; Ringsdorf, H.; Schmidt, F. J.; Knoll, W. Longmuir
-1991. - - -, .7., -1RR7. -- ..
Academic Press: 1987; Vol. 135, pp 3 M . (2) Egger, M.; Heyn, S.P.; Geub, H. E. BBA 1992,1104,45-54. (3)Schuhmann, W.;Heyn, S.P.; Gaub, H. E. Adu. Mater. 1991, 3, 388-391.
0743-746319312409-0136$04.00/0
(0) Fischer, B.Diplomarbeit Theaie, Technischen Univmitllt Mhchen,
198s.
(7) Theriault., T. P.; Leahy, D. J.; Levitt, M.; McConneU, H.M.; Rule, G. S.J. Mol. B801. 1991,221, 257-270. (9
1993 American Chemical Society
Antigen Binding to Lipid- Anchored Antibodies
Langmuir, Vol. 9, No. 1, 1993 137
-
Wet Brldge
A
-
2 nm
Glycerophospholipid L
Langmulr-Blodgett Trough
Control of the Lateral Packlng and Transfer onto Solld Support
Llpld / Proteln Monolayer from a Veslcle Suspenslon
fragment
8nm
Binding Pocket m n 3 nm
m
Figure 1. Scheme of the model receptor used ir, this study. It is a synthetic hybrid molecule consisting of the antigen binding fragment of a monoclonal antibody linked to a phospholipid via a flexible spacer. For brevity we named it Fab-lipid. by pepsin cleavage. The antibody fragments were then bound to a spacer lipid which was presynthesized by reacting dipalmitoylphosphatidylethanolamine(ta-DPPE, Sigma)with the heterobifunctional cross-linker N-succinimidyl 3-(2-pyridyldithio)propionate (Pharmacia/LKB). The coupling reaction between the spacer lipid and labeled antibody fragment was carried out in Hepes buffer (0.01 M Hepes, 150 mM NaC1, pH 7.45). Since the spacer lipid does not form stable vesicles, it was mixed with dimyristoyl-L-a-phosphatidylcholine(DMPC). Small unilamellar vesicles were prepared by sonication for 10 min in couplingbuffer usinga tip sonifier (Branson,IL). Fab' fragments were added to the vesicle solution, and the mixture was kept a t 25 "C for 16 h. A molar ratio of 0.04 of DPPE-DTP to DMPC and a final concentration of protein of 1.2 mg/mL yielded the highest amount of Fab-lipid; further details are given in ref 6. The resulting Fab vesicles were finally purified either by gel filtration on Sepharose CL 4B or by density gradient centrifugation in a metrizamid (Serva, Heidelberg, FRG) gradient. Binding Assay. Bovine serum albumin (BSA) was haptenated with 2,4-dinitrophenol (DNP) (Sigma) following the protocol in ref 2. The labeling resulted typically in 5-8 DNP groups per BSA. For the binding assay the DNP-BSA was dissolved at a final concentration of 10 nM in blood serum (BayrischersRotes Kreuz). For secondary labeling of the antiDNP antibodies,polyclonal goat antimouse antibodies were used (Sigma). Preparationof Planar SupportedFab-Lipid Monolayers. The preparation of Fab-lipid monolayers from a vesicle suspension and the subsequent monolayer transfer onto a solid support were performed with a miniaturized microfluorescence film balance that has been described in detail in ref 8. This rather unusual way of spreading a monolayer was necessary because organic solvents, which are usually used, denature the proteins. The setup shown schematicallyin Figure 2 consists of a miniature Langmuir trough and a temperature-controlled well where vesicles spread a monomolecular film at the air-water interface. A thin film of buffer on a strip of filter paper bridges the two compartments and allows the lipid-protein monolayer to expand onto the surface of the Langmuir trough subphase while the vesicles are retained in the spreading well. For details of the spreading method see ref 9. After removal of the wet bridge the Fab-lipid monolayer was compressedin the Langmuir trough until a phase separation into protein-rich domains and (8) Heyn, S. P.; Tillmann, R. W.; Egger, M.; Gaub, H. E. J. Biochem. Biophys. Methods 1990,22,145-158. (9) Heyn, S . P.; Egger, M.; Gaub, H. E. J. Phys. Chem. 1990,94,50735078.
Figure 2. Experimental setup for the formation of the recognition pattern. In the small temperature-controlled trough on the left side, a monolayer is formed in equilibriumwith the vesicle phase. By increasing the surface pressure this monolayer is pushed via a wet bridge onto the surface of the adjacent Langmuir trough where it is compressed and transferred.
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Figure 3. Calculated surface plasmon resonances for two dielectric films with different thicknesses. The calculation is basicallya numericsolutionof the Fresnelequation of a multilayer system. The parameters were 500-A gold film (e = -12.02 + i1.05) on a 60" prism (n = 1.723) and a dielectric film (n = 1.5) with a 20-81or 60-A thickness in water (n= 1.33). The excitation wavelength was = 632.8 nm. The dashed lines indicate the angles a t which Figure 6 was taken. a phase of mainly pure DMPC was obtained.'') This monolayer was transferred by the horizontal dipping technique onto one side of a high refractive index glass prism which was coated with a 500-81 gold layer. The hydrophobic gold layer exhibited a forward contact angle toward pure water of typically 7 ~ =~80". 0 Alternatively the gold film was precoated with a self-assembled alkyl chain film as given by Whitesides et al.ll After the prism had been carefully pushed through the air-water interface (see Figure 2) the resulting LB film was always kept submerged by means of a glass slide with two attached spacers that formed a microchamber covering the prism. This chambercould be flushed and incubated while performing SPM measurements. Surface Plasmon Microscopy. Surface plasmons are electromagnetic waves which propagate with an exponentially decaying penetration depth at the interface between a dielectric and a metal. Surface plasmons are excited by p-polarized electromagneticwaves (in our case by visible light) provided the dispersion relations of both waves are matched. Thus, the dispersion relation may be probed by varying the incidence angle of the excitingwave (and by means of this the momentum transfer parallel to the interface) a t a fixed energy. When the dispersion relation is crossed, the resonant excitation of plasmons will lead to a sharp decrease in reflectivity (see Figure 3). This resonance is extremely sensitive to variations in the refractive index of the dielectric near the interface. When illuminated in resonance conditions for a certain dielectric layer, e.g., a homogeneous lipid (10) Egger, M.; Ohnesorge, F.; Weisenhom, A.; Heyn, S. P.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P.; Gaub, H. E. J. Struct. Biol. 1990,103,89-94. (11) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.;Eval, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. SOC.1989,111,321-335.
138 Langmuir, Vol. 9, No.1, 1993
P Xe
Kdhler Optics
Fischer et at.
Camera
Image Analyzer
Arc Lamp
Figure 4. Scheme of the surface plasmon microscope. film, virtually no light is reflected from the interface. However, a local change in the thickness of the film, e.g., by bound macromolecules, leads to a drastic increase in the brightness of this spot when seen through suitable optics. Thus, it is obvious that the surface plasmon resonance can be used for contrast enhancement in microscopy. Rothenhaeusler et al.I2 have shown this for the first time. The lateral resolution of this technique is determined by the propagation length of the plasmons which is inversely proportional to the width of the resonance. For gold in water and an excitation in the visible range, the resolution limit is on the order of 5 pm.6 This means that the requirements for the aperture of the microscope optics are low; the setup can be simple. The scheme of our surface plasmon microscope is given in Figure 4. A parallel illumination beam is made from the light of a xenon arc lamp by KGhler optics. The beam passes a line filter (A = 632 f 5 nm) and a linear polarizer before it hits the high index prism at an adjustable external angle Y ~ .An image of the gold film is projected onto a CCD camera which is tilted accordingly to compensate for the tilt of the gold film with respect to the optical axis. The optical magnification is 10-fold,resulting in a resolution on the CCD of 1pm/pixel with a field of view of 500 pm. Surface plasmon images (taken under p-polarized illumination) are transferred via a frame grabber (Leutron vision, Germering FRG) to a computer (IBM-AT) where they are normalized by a reference image of the same spot which was taken with s-polarized illumination. An image, taken in s-polarized illumination, contains no plasmonic contributions and basically reflects the illumination profile and inhomogeneities in the film. Unfortunately in our SPM the incidence angle could not be easily varied during an experiment. For monitoring binding experiments we therefore abolished spatial resolution and used a linear camera in combination with a wedge illumination similar to the setup given in ref 6.
Results and Discussion As described in the previous sections, we allowed a monolayer containing Fab-lipid to form at the air-water interface, starting from a vesicle suspension. The monolayer was then slowly compressed and simultaneously monitored by quantitative microfluorescence. After the onset of the phase segregation,whose nature is described elsewhere,l3the film was slowly compressed further until a pattern of about equal area coverage (see Figure 5) of the protein-rich phase (bright) and the protein-depleted phase (dark) had formed. The fluorescenceintensities of the homogeneousfilm as well as from the two phases after segregationwere recorded. With the known lipid/protein ratio from the synthesis of the Fab-lipid, the fluorescence intensities were converted into molar ratios and after correction for the area changes converted into average area occupancies(see Table I). For the latter value in the molecular dimensions in Figure 1 were used. The film was then transferred onto a prism and investigated with the SPM. Figure 6 showssuch a phase-segregated film as seen with the SPM. The domain pattern is again clearly (12) Rothenhlusler, B.; Knoll, W. Nature 1988,332,615-617. (13) Egger, M.; Heyn, S.P.; Gaub, H. E.Biophys. J. 1990,57,66!+-673.
Figure 5. Microfluorescence image of the recognition pattern formed at the air-water interface prior to transfer onto the solid support (image size 300 pm). ~~~~~
molar protein content (%I
phase homogeneous 0.5 f 0.1 protein-rich lf0.2 protein-depleted 0.2 f 0.1
Table I . average area occupancy (%) 14 f 3 22f5 4f2
average average refractive thickness index (nm) 1.36 1.38 1.34
1.1 f 0.2 1.8f 0.4 0.4 f 0.2
visible. As can be observed also at the air-water interface, the domains are often-most probably due to lateral flow-arranged in a stripelike pattern. The two images in Figure 6 were taken at the two angles where the contrast between the patches and the background was at a maximum. The incidence angles were at about 56O, and the difference between the two angles, at which the two phases showed minimum reflectance, was A = 0.6 f 0.2'. The high contrast which is clearly inverted in the two pictures shows that surface plasmons can be excited at two discrete angles only. This in turn means that the areas on the film correspondingto the two different phases have two distinct optical thicknesses. If we take the Fablipid content measured by fluorescenceand convert it into either an average refractive index or an average thickness, these two resonances may be simulated in Fresnel calculations. This approach isjustified since the distribution is averaged over the typical decay length of a plasmon which is in the order of 5 pm. With the refractive indices of nprot= 1.55 and = 1.33, a lipid moiety with nlipid = 1.48,and a thickness of 20 A, the resonancesare expected at 56.05' and 56.36O. The measured values are therefore in good agreement with theoretical predictions. They support our model of these recognition patterns which was developed earlier by means of other techniques such as atomic force microscopy (AFM).1°J4 Binding of antigens to the pattern could qualitatively be observed with the SPM. However, in order to be able to continuously monitor the binding of antigens to our receptor surface, we switched to a SP spectrometer. For this experiment the film was kept below the critical pressure for the phase segregation. The film was verified to be homogeneous by fluorescence microscopy before transfer. Figure 7, top, shows the time course of the experiment. As the first step the receptor film was blocked (14) Weisenhom,A.L.;Drake,B.;Prater,C.B.;Gould,S.A.C.;Hanama, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S.P.; Gaub, H.E. Biophys. J. 1990, 58,1251-1258.
Antigen Binding to Lipid- Anchored Antibodies
Langmuir, VoZ.9,No. 1, 1993 139
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0
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,30
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Time [minutes] Anti Mouse Antibody AN02
Figure 7. (Top) time course of a binding experiment to a Fablipid-containingfilm measured with surface plasmons. (Bottom) Scheme of the molecularlayer built up during the different steps of the experiment in the top panel.
Figure 6. Recognitionpattern formed from a phase-segregated Fab-lipid film as seen with the SPM a t two different excitation angles (image size 200 pm): (top) 54O, (bottom) 57'.
against nonspecific binding by incubating with a solution of 3% BSA in Hepes for about 5 min. After the chamber was flushed with Hepes, the new resonance angle was measured. Blocking obviously changed only little of the surface coverage. Then the film was incubated with a sample of full blood serum containing haptenated BSA as the test antigen at a concentrationof 10nM. After flushing with Hepes, the measured shift in the plasmon resonance was only minor. After incubation with a 1pM solution of AN02, an antibody against the hapten, and subsequent flushing,the resonancewas markedly shifted. Subsequent incubation with a polyclonal antimouse antibody lead to a further slight shift in the resonance. The width of the resonance did not significantly change throughout the experiment. For each incubation step the change in the effective thickness was calculated from the measured change in the resonance angle using again Fresnel simulation of the multilayer system (Figure 7). The refractive index of the bound protein was also taken to be n& = 1.55. The effective thickness changes of the film upon addition of antigen and the primary antibodies agree well with the expectedvalues,taking into accountthe sparsedistribution of the receptors (see Table I) and the multivalency of the antigen. The sparse distribution makes it also plausible that the nonspecific adsorption of the blocker resulted in a thickness change, which corresponds to roughly half the amplitude of the first specific binding. The blocked receptor surface was obviously not subject to further
nonspecific adsorption from the blood serum. The large antibodies that sandwich the multivalent antigen then lead to the most significant signal. This is a well known effect which is used in standard immunoassays. The incubation with the secondary antibody, however, did not result in a comparable thickness increase. This might be caused by the poor binding constant of the polyclonal antibody for this particular mouse antibody. This picture is schematically summarized in Figure 7, bottom. These results demonstrate the potential use of such receptor films in combination with surface-sensitive optics in immunoassays. Concluding Remarks Our experiments showed that lipid films with our custom-designed lipid-anchored receptors containing the binding site of a monoclonal antibody may be transferred onto a solid support such that the functionality of the receptor is maintained. At the air-water interface these films may be driven into a phase segregation,resulting in a receptor pattern. After transfer onto a solid support such films may be used as a recognition pattern for certain antigens. We visualizedthis pattern with surfaceplasmon microscopy, and we characterized its optical properties. The use of surface plasmons allowed us to quantify the amount of bound antigen. An improved design of our surface plasmon micr0scope,~5which would allow the online scan of the incidence angle while recording images, should greatly improve the sensitivity of an assay based on such laterally structured receptor films. Furthermore, extremely sensitive measurements are conceivable if one also uses changes in the imaginary part of the refractive ~
~~
(15)Schmidt, F. J.; Knoll, W.Biophys. J. 1991,60,716-720.
140 Langmuir, Vol. 9, No.1, 1993
index which might be cawed by some appropriate enzymatic activity initiated by the binding event.
Acknowledgment. The cell line that produced the monoclonal antibody AN02 was a kind gift from Harden McConnell. We are indebted to Benno Rothenhiiualer
Fiacher et al. for helpful discussions. We would like to thank Karen Mason for carefully reading thh paper. Thin work was supported by the Stiftung Volkswagenwerk and the Deutache Forachung6gemeinachaft. Registry No.
DMPC,18194-24-6.
