Langmuir 1996, 12, 4857-4865
4857
Scanning Angle Reflectometry Study of the Structure of Antigen-Antibody Layers Adsorbed on Silica Surfaces L. Heinrich,† E. K. Mann,*,†,‡ J. C. Voegel,‡ G. J. M. Koper,§ and P. Schaaf†,| Institut Charles Sadron (CNRS-ULP), 6, rue Boussingault, 67083 Strasbourg Cedex, France, Institut National de la Sante´ de la Recherche Me´ dicale, Unite´ 424, Centre de Recherches Odontologiques, Universite´ Louis Pasteur, 1 place de l’Hoˆ pital, 67000 Strasbourg, France, Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and Ecole Europe´ enne de Chimie, Polyme` res, et Mate´ riaux., B.P. 296F, 1, rue Blaise Pascal, 67008 Strasbourg Cedex, France Received March 18, 1996. In Final Form: July 2, 1996X A layer of Immunoglobulin G (IgG) adsorbed on silica is brought into contact with a solution of IgG antibodies that specifically bind to the first protein. Both the thickness and the density of the resulting layer are studied using scanning angle reflectometry. Initially, the incoming antibodies appear to embed themselves in the preadsorbed protein layer. The structure of the layer relaxes with time, becoming both thicker and less dense. This evolution in structure continues long after the total surface concentration stops evolving. The final protein layer is surprisingly thick: as much as three times the length of a single protein molecule. We hypothesize the formation on the surface of an antigen/antibody complex that subsequently turns there, as previously postulated for the exchange process. This would expose the antigen to further interactions with antibodies in solution, allowing the formation of a triple layer.
I. Introduction The adsorption of proteins on solid surfaces has received considerable attention over the last 2 decades, due to its importance in the food and drug industry and in the understanding of, for example, biocompatibility. The first studies in this field focused on the determination of adsorption isotherms.1 However, it became rapidly clear that one of the characteristics of the adsorption of macromolecules, and in particular of proteins, is its partially irreversible character: when a solid surface covered with macromolecules is placed in contact with pure buffer or solvent, the macromolecules often remain adsorbed.2 Surprisingly, when such a layer is brought into contact with a solution of macromolecules (different from or identical to the adsorbed ones), a large number of adsorbed molecules can be replaced on the surface, in what is called an exchange process.3 Kinetic laws have been proposed for the exchange of synthetic polymers4 and for proteins.5,6 However, the mechanism is not yet understood from a microscopic view. It may involve gradual replacement of one molecule by another; the formation of a complex between the adsorbed protein and a protein in the bulk, followed by an inversion of the complex to place the incoming protein on the surface, has also been suggested.7 However, such inversion of two proteins on a surface has never been demonstrated. * To whom correspondence should be addressed at Institut Charles Sadron. † Institut Charles Sadron. ‡ Institut National de la Sante ´ de la Recherche Me´dicale. § Leiden Institute of Chemistry. | Ecole Europe ´ enne de Chimie, Polyme`res, et Materiaux. X Abstract published in Advance ACS Abstracts, September 15, 1996. (1) McRitchie, F. Adv. Protein Chem. 1978, 32, 283. (2) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (3) Brash, J. L.; Uniyal, C.; Pusineri, C.; Schmitt, A. J. Colloid Interface Sci. 1983, 95, 28. (4) Pefferkorn, E.; Carroy, A.; Varoqui, R. J. Polym. Sci. 1985, 23, 1997. (5) Huetz, Ph.; Ball, V.; Voegel, J.-C.; Schaaf, P. Langmuir 1995, 11, 3145. (6) Ball,V.; Huetz, Ph.; Elaissari, A.; Cazenave, J.-P.; Voegel, J.-C.; Schaaf, P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7330. (7) De´jardin, P.; Le, M.-T. Langmuir 1995, 11, 4008.
S0743-7463(96)00263-6 CCC: $12.00
These studies show (i) that the thermodynamic concept of isotherm, valid for the adsorption of small molecules on solid surfaces, does not truly apply to the adsorption of macromolecules, and (ii) that in spite of the nearly irreversible nature of the adsorption process, the adsorbed proteins do not behave like static entities, but rather show many important dynamic aspects, which are poorly understood. This dynamic character shows up in the denaturation process of adsorbed proteins on surfaces,8 in the restructuring of adsorbed layers with time, and finally in the exchange process. On the other hand, studies performed by means of optical techniques, which are designed to investigate the structure of the adsorbed layer, demonstrate that when a protein solution is in contact with a solid surface, the proteins adsorbing on the surface usually form a single layer;9 only rarely is there multilayer formation. For example, as IgG molecules adsorb, the layer thickness remains constant while the refractive index increases with time,9 typical of the building up of a simple monolayer. However, if the protein is not spherically symmetric, the structure of this layer can be complex, as suggested from adsorption studies of fibrinogen on silica by means of scanning angle reflectometry (SAR)10 or by ellipsometry;9 here, thickness may increase with time, as the proportion of molecules adsorbed in different configurations, for example, “side-on” and “end-on”, changes. Despite the widespread use of optical techniques to investigate the structure of adsorbed layers, very few results are reported concerning the “optical kinetics” in which one follows the evolution of the optical thickness and the mean refractive index. In this article we use SAR to investigate the antigenantibody reaction when the antigens are already adsorbed on a solid surface. We will analyze the structure of the layer of proteins thus formed and demonstrate a longtime evolution of this structure. Naively, one would expect that when a layer formed by preadsorbed antigens is brought into contact with a solution of its antibodies, a layer of the antibodies forms on top of the antigen layer, (8) Ball,A.; Jones, A. L. Langmuir 1995, 11, 3542. (9) Malmsten, M. J. Colloid Interface Sci. 1994, 166, 333. (10) Schaaf, P.; De´jardin, Ph.; Schmitt, A. Langmuir 1987, 3, 1131.
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provided the conformation of the adsorbed antigen is such as to allow the antigen-antibody reaction to take place. We demonstrate that while such reactions occur, the layer which is built up is very different from this expectation and may reach three times the thickness of a single protein layer. Furthermore, the structure evolves over time scales which are long compared to the characteristic time over which the adsorbed material increases. This study is thus complementary to the ones devoted to the desorption or the exchange process, in demonstrating the dynamic nature of the adsorbed layers. II. Material and Methods A. Proteins. Both proteins belong to the immunoglobulin G (IgG) family of antibodies. The first is a human IgG (called “antigen” or “I” in what follows, in order to readily distinguish the two molecules). The second is a rabbit IgG (called “antibody” or “aI” here) produced in an immune reaction against the human IgG antigen. The proteins are thus very similar in size and shape (see Table 1). The difference lies in where and how they bind to each other. IgG molecules are roughly Y-shaped;11 at the top of each of the two arms of the Y there is an active domain, able to fix an antigen. Here, I acts as an antigen to the aI antibody. A given active region reacts with one particular molecular pattern on the antigen. However, our antibody sample is polyclonal: different molecules in the sample fix on different parts of the antigen. We thus see that in our case the antigen/antibody interaction is asymmetric: only two regions on the antibody molecules play a role in binding, while many sites on the antigen may be bound to some antibody (see Table 1 for a summary of the characteristics and notation for the different protein molecules.) Proteins were received in powder form from the Centre Re´gional de Transfusions Sanguines of Strasbourg and were dissolved in a phosphate buffer (PBS), made by adding an acidic solution (50 mM NaH2PO4‚H2O and 0.15 M NaCl) to a basic one (50 mM Na2HPO4‚7H2O and 0.15 M NaCl) until a pH ) 7.5 is reached. Protein solutions were kept frozen in small 1 mL aliquots, at concentrations of about 3 g/L for the antigen samples and 5 g/L for the antibody samples. The concentrations were determined by adsorbance measurements using a UV spectrophotometer (Beckman model DL 640, wavelength 280 nm). For all these experiments, the final protein solution was prepared less than 1 h before introduction into the measurement cell: the aliquots were immersed in a bath between 30 and 40 °C for a few minutes, just until the protein solution was nearly melted. Then, the solution was added to more PBS to achieve the desired protein concentration. Both buffer and protein solutions were filtered with a Millex GV filter with a pore size of 0.22 µm. Before each experiment, the sample cells were cleaned over a period of about 2 days in a laboratory detergent, Hellmanex II (Helma GMBA, D-79379 Mullheim), at 3%, followed by copious rinsing in pure water and several minutes in a sulfuric acid bath (Prolabo; quality “for analysis”, diluted to 1%). Finally, everything was rinsed several more times with pure water. Deionized SuperQ-Millipore water was used throughout. B. Scanning Angle Reflectometry. In order to analyze the evolution of the structure of the adsorbed layers during the adsorption process of antigens onto solid surfaces and during the antigen/antibody reaction, we used an optical technique, scanning angle reflectometry (SAR). The advantage of using SAR instead of fixed-angle reflectometry or ellipsometry is that the thickness and the surface concentration of the adsorbed layer can be determined independently. The technique has been extensively described in previous articles10,12 and we present it only briefly here. In SAR one measures the reflection coefficient of an electromagnetic wave polarized in the plane of incidence (p-wave) for various incidence angles around the Brewster angle. For a perfect, flat (or Fresnel) interface characterized by an abrupt (11) Bagchi, P.; Birnbaum, S. M. J. Colloid Interface Sci. 1981, 83, 460. (12) Schaaf, P.; De´jardin, Ph.; Schmitt, A. Rev. Phys. Appl. 1986, 21, 741.
Figure 1. Reflectivity as a function of incident angle θ on a silica/buffer solution interface: (o) without protein; (•) after 4 h in contact with a solution of I at 0.2 g/L; (∆) after 60 h of the I-covered surface in contact with a solution of aI at 0.05 g/L. Dotted line corresponds to a fit of the data to the reflectivity of a Fresnel interface, given by eq A1. Solid lines correspond to fits to the reflectivity of a uniform homogeneous layer, given by eq A2. change of the refractive index between the two media, the reflection coefficient is zero at the Brewster angle. At this particular angle, the direction of propagation of the transmitted wave is perpendicular to that of the reflected wave. The molecular dipoles excited in the transmitted medium are parallel to the direction of propagation of the reflected wave and thus do not radiate any energy in the direction of coherent scattering that constitutes the reflected beam. Now consider a layer of adsorbed molecules at the interface. The refractive index of this layer is in general different from that of both the incident and the transmitted medium. In this layer, the refracted beam is no longer perpendicular to the direction of propagation of the reflected wave and the excited dipoles from this layer radiate energy in the direction of reflection; the reflection coefficient is no longer zero. One can show that not only does the reflected intensity at the Brewster angle differ from zero in the presence of an adsorbed layer but the whole reflectivity curve (reflected intensity as a function of the incidence angle) changes shape and position (see Figure 1). It is these variations in the reflectivity curve that are analyzed to extract information about the adsorbed layer. For protein layers, which are generally much thinner than the wavelength of light, one can mainly determine a mean thickness and the mean refractive index of the layer. The fact that for a Fresnel interface the reflection coefficient vanishes at the Brewster angle leads to a high sensitivity to adsorbed layers. The light source of the reflectometer is a 5 mW HeNe laser (λ ) 632.8 nm). The beam is polarized before entering nearly perpendicularly through one face of a prism. It reflects then on the hypotenuse of the prism which is in contact with a buffer solution. This face constitutes the adsorption surface and is optically flat to λ/4, where λ corresponds to the wavelength of light in the vacuum. The reflected beam then leaves through the third face of the prism, again nearly perpendicularly, before it passes through a second polarizer. This second polarization is required because we will measure reflection coefficients of the order of 10-7, so that s-polarization, with its high reflectivity, must be eliminated to this degree. The intensity is then measured by means of a photomultiplier. The angles of incidence are selected by rotating the cell (and thus the interface) by means of a high-precision (0.001°) goniometer (Microcontrole, Evry, France); the position of the photodetector is adjusted to find the position of greatest intensity with the same precision. Any residual s-component of the light was considered as part of a constant background, so that the intensity was assumed to follow the law I(θ) ) I0 + ARp(θ), where Rp corresponds to the intensity reflection coefficient for p waves, A is an instrumentdependent constant, and I0 represents the residual signal at the Brewster angle. I0 was typically an order of magnitude less than the minimum intensity for the thinnest protein layers and 3 orders of magnitude less than the maximum intensity measured. Strictly speaking, the residual s-component of the reflected light, Rs, will also depend on the angle, so that I0 would not be strictly
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constant. However, for the angular range explored, Rs varies by only 10%, and even if I0 was entirely due to this Rs, the error introduced by ignoring its angular dependence would be everywhere more than 2 orders of magnitude less than the purely statistical noise level; taking I0 as constant had no effect on the results of the curve-fitting procedure. The experiment was conducted as follows: The reflected intensity was first measured for a pure buffer-silica interface, with a Brewster angle of approximately 42.5°, at discrete incidence angles 0.02° apart, ranging typically from 41.5° to 43.5°. This curve was compared to the Fresnel reflection law, which allowed for a calibration of the reflectometer. The antigen solution was then rapidly injected into the cell. The adsorption of the proteins on the silica surface was followed by measuring reflectivity curves at repeated time intervals. The determination of each reflectivity curve took approximately 7 min and the minimum time between the measurement of two curves was also approximately 7 min. During the adsorption process, the solution was at rest in the cell so that only the diffusion process of the molecules in the solution and the interaction mechanism between the proteins and the surface influenced the adsorption kinetics. In order to stop the adsorption process and to replace the antigen solution in the cell by an antibody solution, the cell was first flushed with pure buffer, which also removed any loosely adsorbed antigen molecules. This flushing of the cell was very extensive, with about 100 times the volume of the cell over a 12 h period, in order to minimize the antigen concentration in the cell before antibody molecules were injected; otherwise these two kinds of molecules would have interacted one with another in the solution and no longer only on the surface. After this flushing step, an antibody solution was injected into the cell and the reaction of the antibody molecules with the adsorbed antigen molecules was again followed by determining reflectivity curves as a function of time. To determine the parameters characterizing the silica-protein interface, a given refractive index profile in the direction normal to the interface needs to be assumed. The simplest and most common model consists of approximating the protein layer as homogeneous and isotropic. The layer is thus characterized by a mean refractive index increment ∆n (the difference between the refractive index of the film n and of the solution, nw) and a mean thickness L. For such a simple situation, an analytical expression of the reflection coefficient Rth(θi;∆n,L) exists (see Appendix 1). The parameters L and ∆n are determined by a nonlinear least-squares regression procedure fitting Rth(θi;∆n,L) to the experimental values Rexp(θi). In order to obtain both parameters with sufficient precision, it is necessary to have accurate data for many independent angles (here about 50) over a large angular range. Figure 2 shows the evolution of the quality function χ2 as a function of the two fitting parameters, ∆n and L, for a typical experimental curve. The quality function is the standard least-squares parameter
Figure 2. Contours of constant quality function χ2, as defined in eq 1, for the fits of the reflectivity of a protein layer to the model of a uniform homogeneous film (eq A2), varying the two parameters of the model, the thickness of the film L, and the refractive index increment ∆n. The reflectivity curve, corresponding to intermediate coverages, was taken after 33 h of contact between the antigen-covered surface and an antibody solution at a concentration of 0.1 g/L. The parameters corresponding to the best fits for this curve are L ) 30.5 nm, ∆n ) 0.0300, and χ2 ) 0.177, shown as a point at the center of the graph, surrounded by contours for which χ2 ) 0.5, 1, 5, 10, 100, and 300 successively. estimate of the surface concentration by only 1%, which is insignificant here. We also assume that dn/dc is not significantly affected by any conformational changes that the protein might undergo during adsorption, which is reasonable given that dn/dc varies little between different kinds of proteins. More importantly, we assume that while there may be conformational changes or orientation of the molecule at the surface, these do not induce any significant optical anisotropy between the normal and the in-plane directions. Since protein molecules are generally quite amorphous,15 this is at least plausible, unless there is formation of a two-dimensional crystal within the plane which would be unexpected with irreversible adsorption. We have thus assumed that
(dn dc )
-1
Γ ) L∆n χ2 )
∑ i
[Rexp(θi) - Rth(θi,∆n,L)]2 σ2
(1)
where the expected error σ is estimated to be 0.01Rexp. The shape of the valley around one pair of values for the parameters ∆n and L demonstrates the precision attainable in the determination of these parameters. From these structural parameters, it is also possible to determine the protein surface concentration, assuming that the index of refraction within a protein layer can be approximated by that for a dilute protein solution, ∆n ≡ n - nw ≈ c dn/dc, where c is the (bulk) protein concentration and dn/dc has been found to be of the order of 0.18 g-1 cm3 for a large variety of protein solutions.13 Since the protein may in fact be quite densely packed within the layer, an approximation such as that of Maxwell-Garnett might be more appropriate.14 However, for the very small values of ∆n/nw observed here, typically 0.025, using the more elaborate approximation would change our (13) De Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759. (14) For a discussion of this approximation in the context of protein monolayers, see: Frey, W.; Schief, W. R., Jr.; Vogel, V. Langmuir 1996, 12, 1312.
(2)
taking dn/dc ) 0.18 g-1 cm3. If a better approximation is found in the future, L∆n can be recovered for the new calculation using the simple multiplicative factor; however, we expect the value of Γ calculated using (2) to be good to within 5%. C. Additional Characterization Techniques. We observed surprisingly high values for the thickness of the antigen/ antibody layer. It is thus particularly important to verify the purity of the antibody solutions and in particular possible contamination by large molecules (most likely IgM molecules, also involved in the immune response, which are similar to an IgG pentamer in appearance) or aggregates in the antibody solution. Three independent tests were performed: electrophoresis, light-scattering, and immunoprecipitation. (i) Electrophoresis. Electrophoresis (SDS-PAGE) is a standard tool to test the composition of protein solutions.16 The experiment, performed at the Centre Re´gional de Transfusions (15) This is in contrast with the case of the chain surfactants often studied by optical techniques, where a simple alignment of the chains at the surface can produce an anisotropy of 5%, see for example: Paudler, M.; Ruths, J.; Riegler, H. Langmuir 1992, 8, 184. (16) Hames, B. D., Rickwood, D., Eds. Gel Electrophoresis of Proteins: a Practical Approach; IRL Press Limited: London, Washington, DC, 1981.
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Table 1. Characteristics and Notation for the Protein Molecules
molecule
abbr
Mw (kDa)
human IgG rabbit IgG (polyclonal anti-human IgG)
I aI
150 150
sizea (nm3)
I/aI attachment regions
24 × 10 × 4 24 × 10 × 4
many 2
a Dimensions in the “T” conformation (see ref 11). The molecule is flexible.
Sanguines in Strasbourg, consisted of observing the migration of charged molecules in an electric field, through a polyacrylamide gel (4-15% reticulated). SDS (sodium dodecyl sulfate) was added to assure that protein migration depends only on molecular weight.16 No evidence of molecules larger than the IgG was revealed; any larger molecules certainly represent less than 0.2% of the total. (ii) Immunoprecipitation Test. Electrophoresis in the presence of SDS is insensitive to aggregation. An immunoprecipitation test can provide a first estimation of the “cross reaction”, a nonspecific binding between antibody (aI) molecules. In this test, the protein concentration in a solution after precipitation of large aggregates is compared to that after redispersion of this precipitate, using a spectrophotometer to measure the absorbance at 280 nm. Four samples of antibody solutions at different concentrations, 0.1, 0.2, 0.5, and 1 g/L, are immersed, for 1 h, in a bath at 37 °C and then kept 18 h at 4 °C so that the precipitation can occur, followed by centrifugation at 12 000 rpm. No aggregation is detected except in the most concentrated solution, at 1 g/L, which showed 2% cross reaction. The experiments in reflectometry involved solutions with bulk antibody concentrations CaI < 0.3 g/L. (iii) Light Scattering. Quasi-static light scattering experiments17 are able to provide a test which is also sensitive to smaller aggregates. The experiments yielded an average hydrodynamic radius, which can be compared to the expected dimensions of a single protein molecule. We analyzed four samples containing antibody solutions at various concentrations: 0.1, 0.2, 0.3, 0.4 g/L. The light scattering measurements were performed at θ ) 90° with the sample cell in a toluene bath. The data obtained for these four samples were quite similar. Using the CONTIN method of analysis,17 the number of particles with a given hydrodynamic radius shows a single peak, with the instrumentally-determined width. The average hydrodynamic radius is at most 5-6 nm, consistent with the known dimensions of a single molecule (see Table 1) and inconsistent with a significant number of aggregates.
III. Results and Discussion A. Optical Kinetics for the Adsorption of Antigen Molecules. The adsorption of the initial antigen (I) layer was carried out at a protein concentration of 0.2 g/L, which is sufficient to allow saturation of the surface, in the sense that larger bulk concentrations do not lead to higher steady-state surface concentrations. From the experiments, the evolution of the optical thickness L and of the refractive index increment ∆n as a function of time was determined. This also allowed the determination of the evolution of the two structural parameters as a function of the adsorbed surface concentration Γ, related to L∆n through eq 2. We see, in Figure 3, that the optical thickness of the antigen layer remains almost constant during the whole adsorption process whereas the refractive index increment of the layer increases. This is in accordance with the observation by Malmsten9,18 for the adsorption of IgG molecules on both hydrophobic and hydrophilic surfaces. The measured thickness of the adsorbed layer is of the order of 18-20 nm. The flexibility of the IgG molecule, especially in the central region, and (17) Berne, B.; Pecora, R. Dynamic Light Scattering with applications to Chemistry, Biology and Physics; Wiley Interscience: New York, 1976. (18) Malmsten, M. Colloids Surf. B 1995, 3, 297.
Figure 3. The thickness L (O) and refractive index increment ∆n (b) of the protein film which forms in the presence of a solution of I at 0.2 g/L, as a function of the surface concentration Γ. Note that the thickness remains constant while ∆n increases during adsorption. Table 2. Characteristics of the Adsorbed Protein Layers as Determined by Reflectometry proteina
L (nm)c
∆n
Γ (mg/m2)c
I aI I+I aI + Ib I + aI
18 ( 2 13 ( 3 21 18 ( 3 53
0.023 ( 0.003 0.013 ( 0.005 0.027 0.009 ( 0.005 0.025
2.3 ( 0.4 0.8 ( 0.2 3.1 0.8 ( 0.3 7.4
a Protein solutions added successively, in the order given; the first protein is removed from the sample cell by careful flushing between additions. All values represent surface remaining in contact with the final protein solution. The bulk concentration of the first protein was 0.2 g/L and the bulk concentration of the second protein 0.1 g/L. b CI ) 0.2 g/L. c Error limits represent variations over several experiments where available.
the possibility of many different conformations on the surface make it difficult to predict the conformation and the size of an IgG adsorbed on a surface;19 they are likely to depend on the particular surface as well as on environmental factors such as pH. On both hydrophilic and hydrophobic glass surfaces, Malmsten9,18 finds, by means of ellipsometry, thicknesses in good agreement with ours. Similar thicknesses are also observed for IgG molecules adsorbed on latex particles, using dynamic light scattering to determine a hydrodynamic layer thickness.20 Our value, consistent with the previous findings, corresponds to one of the larger dimensions of the IgG molecules.11 Thus, for such surfaces, the molecules seem to adsorb not “flat” on the surface but rather in an “endon” configuration. The fact that the thickness remains constant while the refractive index increases during the adsorption process seems to indicate that the layer is filled until saturation, without significant structural changes that would lead to a modification of the layer thickness. The latter would be the case if a double layer were formed by addition of material to the top part of the layer.21 The saturation value of Γ, given in Table 2, is of the order of 2 mg/m2, which corresponds roughly to a surface coverage of the order of 25-30%, taking the area covered by a molecule as 35 nm2.11 (19) Roberts, C. J.; Williams, P. M.; Davies, J.; Dawkes, A. C.; Sefton, J.; Edwards, J. C.; Haymes, A. G.; Bestwick, C.; Davies, M. C.; Tendler, S. J. B. Langmuir 1995, 11, 1822. (20) Morrissey, B. W.; Han, C. C. J. Colloid Interface Sci. 1978, 65, 423.
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B. Antigen/Antibody Reaction Kinetics. The initial antigen (I) layer described above is subsequently exposed to a solution of antibody (aI) molecules, which can bind to the antigen molecules. The increase in the total surface concentration ΓaI is assumed to correspond to the binding of the incoming antibody molecules. During the first stages of the reaction, one can analyze the reaction kinetics neglecting blocking effects from antibody molecules already on the surface. One can then compare the initial binding rate (dΓaI/dt)0 with the theoretical rate assuming that all aI molecules reaching the surface bind irreversibly to the adsorbed antigens. In this case the binding rate would be totally governed by the diffusion process of the proteins in the bulk near the interface.22 The theoretical diffusion-controlled binding rate is given by:
(dΓaI/dt)theor ) CaI(D/π(t - t0))1/2
(3)
where D represents the diffusion coefficient of the antibody molecules (expected to be 4 × 10-11 m2/s) and t0 the time at which the antibody solution is injected into the cell. This assumes that at time t ) t0, the protein concentration in the bulk is constant and equal to CaI up to the interface. The validity of this assumption is difficult to assure experimentally. However, it can be shown that even if a depletion layer exists at the interface at the beginning of the experiment, the adsorption rate will follow eq 3 after an initial lag.22 It is also assumed that incoming particles are adsorbed without any energy barrier, yielding the boundary condition CaI ) 0 at the adsorbing surface at any time t of the process. In our case, the surface concentration does follow quite well the (t - t0)1/2 law predicted under these assumptions (see Figure 4). This excludes a significant amount of release of antibodies from the surface once they have reacted, which would lead to a (t - t0) law.22 Following Tanimoto and Kitano,23 and assuming the validity of the boundary conditions given above and thus of eq 3, one can determine the ratio F defined as
F ) (dΓaI/dt)0/(dΓaI/dt)theor
(4)
Since (dΓaI/dt)theor is proportional to t′-1/2 ) (t - t0)-1/2 and the initial experimental binding rate has been shown to follow this same law, F may also be written as
F ) (dΓaI/dt′1/2)0/(dΓaI/dt′1/2)theor
(5)
where (dΓaI/dt′1/2)0 is the initial slope of the curves given in Figure 4. A value of F equal to 1 means that all the antibody molecules that reach the surface react with the adsorbed antigen molecules whereas a value of F that is smaller than 1 demonstrates that the reaction at the surface is only partial. Figure 5 shows the evolution of F with the bulk concentration CaI. F is clearly independent of CaI, with a value of about 0.06. This value is very near that found by Tanimoto and Kitano for a similar system. Clearly not all antibody molecules arriving at the surface react there immediately. Several different effects may contribute to this: (1) Only a fraction of the antibody molecules in the solution may be able to react with the adsorbed antigen molecules; for example the antibody molecules which are reactive to the area of the antigen molecule that is in contact with the silica surface would not bind to that (21) Schaaf, P.; De´jardin, Ph. Colloids Surf. 1988, 31, 89. (22) Goodrich, F. C. J. Chem. Phys. 1954, 22, 588. (23) Tanimoto, S.; Kitano, H. Langmuir 1993, 9, 1315.
Figure 4. Evolution of the protein surface concentration Γ with time, after injection of the aI solution at time t ) t0. The aI solution has bulk concentration: (O) 0.03 g/L; (b) 0.05 g/L. The solid lines are fits of the initial adsorption curve (Γe half the saturation value) to the form (t - t0)1/2, expected if diffusion of the molecules to the interface is the rate-limiting step.
Figure 5. Ratio F (defined in eq 5) of the initial experimental adsorption rate and that expected if the rate-limiting step for adsorption is the diffusion of the molecules to the interface, assuming a diffusion constant D ) 4 × 10-7 cm2/s, as a function of the bulk protein concentration CaI.
molecule. This is illustrated by experiments which were performed by adsorbing initially an antibody protein layer on the silica surface. When this layer was brought in contact with a solution of antigen molecules, no reaction was detected. Only the two ends of the Y-shaped antibody (aI) molecules can react, and if these are consistently pointing toward the surface, the reaction becomes impossible due to steric effects. If such considerations apply, the concentration that should be considered in the analysis of the adsorption rates is not CaI but xCaI, where x is the fraction of molecules in the solution suitable to react with the adsorbed layer. If this were the only effect, one could deduce x ) 1/20 in our case. (2) The binding site on the antibody molecule must be properly oriented with respect to the antigen molecule on the surface, even when the binding site on the antigen molecule is accessible; only a small fraction of incoming molecules would have the proper orientation. In bulk, taking into account the tolerance for geometric alignment would predict F ) 10-6.24 Experimentally, the value is closer to 10-4.23 Recent Brown(24) Northrup, S. H.; Erickson, H. P. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3338.
4862 Langmuir, Vol. 12, No. 20, 1996
ian simulations of this process, which include the rotational diffusion of the molecules, are in agreement with these experimental values.22 It would be interesting to perform similar simulations for the case in which one of the entities is fixed on the surface. (3) An energy barrier exists for the adsorption. This case has been extensively studied;22 t1/2 behavior would be observed only at times sufficiently long that diffusion dominates, where all the previous discussion holds. C. Influence of the Antigen/Antibody Reaction on the Structure of the Adsorbed Layer. Next we examined the structure of the layers which result from the binding between the preadsorbed antigen (I) molecules and the antibodies (aI) protein molecules in solution. The reflectivity curves were again analyzed using the isotropic homogeneous layer model. Other models could be imagined: for example, if the antibody molecules simply attach on top of the antigen layer, a bilayer model would be more appropriate. Only three independent parameters can be deduced from experimental reflectivity curves, while a general bilayer model has four.25 Some additional assumptions about the model are required. We tested two such models: one with equal thicknesses for the two layers, the other with the thickness of the bottom layer fixed at the value found for the initial antigen layer. A leastsquares regression procedure was used to determine the best-fits to the refractive indices of the two layers and the remaining characteristic thickness. Both the surface concentration Γ and the overall thickness of the bilayer were, for both models, very close to that found assuming a homogeneous layer.25 The model of a single homogeneous layer is therefore used in the following, so that the entire protein layer, with both antigen and antibody molecules, is characterized by a single thickness L and a mean refractive index increment ∆n between the layer and the solution in the cell. These optical parameters were followed during the formation of an antigen/antibody layer, when the antigen (I) layer described above is exposed to antibody (aI) solutions of different bulk concentrations. Figure 6 shows the typical evolution of the different parameters L, ∆n, and Γ as a function of time. A similar experiment was carried out by introducing antigen molecules instead of antibody molecules in the cell above the adsorbed antigen layer. We observed a slight increase in the refractive index of the layer with the thickness remaining essentially constant (see Table 2). The changes of the structural parameters characterizing the protein layer observed after the introduction of the antibody molecules in the cell in the presence of an antigen layer are thus almost entirely due to the specific interaction between the antigen and antibody molecules. During the antigen/antibody reaction taking place at the surface, the thickness initially increases rapidly and then slowly stabilizes at a value which is a function of the concentration CaI of antibody (aI) molecules in solution. The evolution of stabilized values of the thickness L with CaI is represented in Figure 7a. On the other hand, the refractive index increment of the layer increases during the first stage of the adsorption process, reaches a maximum, and then decreases with time to a value which is almost equal to the refractive index increment of the initial antigen layer, slightly higher for very high CaI values (see Figures 6 and 7b). Finally, the antibody surface concentration increases rapidly with time before reaching a constant value which is a function of CaI (see Figures 6 and 7c). Since the antigen molecules remain on the surface and antibody molecules remain in the solution for (25) Heinrich, L.; Mann, E. K.; Voegel, J.-C.; Schaaf, P. In preparation.
Heinrich et al.
Figure 6. Thickness L, refractive index increment ∆n, and surface concentration Γ of an I/aI film as a function of the contact time t between the surface and the protein solutions. The protein solution in contact with the surface is as follows: I at 0.2 g/L to the left of the dashed line on the graphs, followed by flushing the cell to remove the I solution, beginning as indicated by the arrow; aI at 0.05 g/L to the right of the dashed lines on the graphs.
more than 20 h, one reason for this saturation could have been the denaturation of the antigen or the antibody molecules before adsorption can take place. We have verified that this is not the case: An antibody solution (0.1 g/L), left at room temperature for a period (20 h) similar to the characteristic saturation time, before being injected into the cell, still reacted with the antigen-covered surface. Further, antigen molecules left on the surface for a period (40 h in all) again similar to the characteristic saturation time also continued to react with fresh injected antibody molecules. This leaves open the question of why saturation takes place at protein surface concentrations less than the maximum absorbable amount; similar saturation of the surface concentration, varying with bulk concentration gives the so-called protein adsorption “isotherm”. Proposed explanations for the latter include concentration-dependent steric hindrance.26 The maximum in ∆n is reached while the thickness of the layer and the protein surface concentration are still increasing. Further, both ∆n and L continue to evolve after Γ has essentially saturated (see also Figure 8). This clearly demonstrates that during the antigen/antibody reaction on the surface, the layer of proteins reorganizes itself over a characteristic time which may be as long as 3 days whereas the reaction takes place much more rapidly. The fact that the refractive index increment first increases with time means that the layer becomes on average denser during the initial stage of the antigen/ antibody reaction; this suggests that initially the incoming antibody molecules are partially embedded in the antigen layer, as suggested in the drawing in Figure 9a. During (26) Ramsden, J. J. Chem. Soc. Rev. 1995, 24, 73.
Antigen-Antibody Layers
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Figure 7. Parameters at saturation for an I/aI film, as a function of the concentration CaI of aI in solution: (O) values for I alone, before addition of the aI solution; (b) values after contact of the I layer with the aI solution. The initial I film was formed by contact of the surface with a solution of I at 0.2 g/L, followed by flushing to remove the I solution. The film is characterized by (a) the thickness L, (b) the refractive index increment ∆n, (c) the surface concentration Γ, and (d) the ratio of the number of aI in the layer to the number of I in the initial layer ((ΓaI/I - ΓI)/ΓI, since the two molecules have the same molecular weight).
the second stage of the process, the structure of the layer changes so as to render the layer both thicker and less dense. The time scale associated with this reorganization process is of the order of 1 to 2 days. It is thus extremely slow and moreover of the same order of magnitude as the characteristic desorption time, estimated to be 44 h, of IgG molecules adsorbed on silica as observed by radiolabeling techniques.5 The thickness of the combined protein layer reaches a saturation value which is of the order of 60 nm at high CaI. This corresponds roughly to a thickness of about 3 times the largest dimension of the proteins. The surface concentration also saturates at high bulk concentrations, at a value about 5 times the original antigen surface concentration, implying that up to roughly four antibodies can associate with each antigen (see Figure 7d). Similar results were obtained using ellipsometry,27 with up to 2.5 antibody molecules per antigen molecule when the initial adsorption takes place on a silicon oxide surface, and a larger antibody/antigen ratio of 5 on a silanated surface. On a gold surface, ratios of about 1.5 were found using the surface plasmon resonance technique.28 Neither of these (27) Duschl, C.; Hall, E. A. H. J. Colloid Interface Sci. 1991, 144, 368. Note that these authors express their data in terms of a layer thickness but that they are obliged to assume a protein refractive index; usually, ellipsometry of thin dielectric films is only sensitive to L∆n, which is more legitimately a surface concentration, as seen in eq 2. (28) Geddes, N. J.; Martin, A. S.; Caruso, F.; Urquhart, R. S.; Furlong, D. N.; Sambles, J. R.; Than, K. A.; Edgar, J. A. J. Immunol. Methods 1994, 175, 149.
studies were able to determine the thickness of the protein layer. The large antibody/antigen ratios may be explained by the polyclonal nature of the antibody molecules. One may also suspect the presence of either larger protein molecules or antibody aggregates in the solution, interacting with the antigen molecules adsorbed on the surface. Two types of experiments have been performed to test the latter possibility: (1) direct tests of the presence of large molecules and of aggregates in the antibody solution, using electrophoresis, light scattering, and immunoprecipitation and (2) inverting the protocol for the reflectometry experiments, first adsorbing the antibody molecules on the surface, flushing well, and then introducing the antigen solution to interact with these. Aggregates in the initial antibody solution in quantity significant for these experiments should show up in the thickness of the initial antibody layer, remembering that the antigen and antibody molecules are the same size. The light scattering and other direct experiments suggest that if aggregates are present in the solution, their concentration is very small (see section II). In the SAR experiments, the initially adsorbed antibody layer had a thickness L and a refractive index increment ∆n of the order of 13 nm and 0.01, respectively (see Table 2). This result strongly suggests that few if any antibody aggregates are present in the solution. After adsorption of antibody (aI) molecules, the solution in the cell was replaced by an antigen (I) solution; no evolution of the thickness and of ∆n was observed (see Figure 10). This
4864 Langmuir, Vol. 12, No. 20, 1996
Figure 8. Characteristics of an I/aI film in function of the surface concentration Γ, for CaI: (b) 0.01g/L; (O) 0.03 g/L; (4) 0.15 g/L. The parameters are (a) the film thickness L; (b) the film refractive index increment ∆n. Arrows indicate increasing time.
a
b
Figure 9. Hypotheses for the I/aI layer structure, consistent with the kinetics and the layer thicknesses observed in this study. Solid figures represent the I molecules, open figures represent the aI molecules, with solid portions representing the regions which specifically bind to the I molecules. Note that the aI sample is polyclonal; i.e., different aI molecules may bind to different regions of an I molecule. (a) At early times, the aI molecules may be embedded in the initial I layer. (b) A sandwich model, yielding a thickness for the protein layer which is roughly three times the molecular size, as observed after long adsorption times. This model implies a slow rearrangement within the protein layer.
surprising result can be explained by the asymmetry of the antigen/antibody reaction: only a limited number of antibody sites can react with the antigen so that if the antibody is oriented on the surface so as to hide these
Heinrich et al.
Figure 10. Thickness L, refractive index increment ∆n, and surface concentration Γ of an aI/I film as a function of the contact time t between the surface and the protein solutions. The protein solution in contact with the surface is as follows: aI at 0.2 g/L in section 1 on the graph, followed by flushing the cell to remove the aI solution, beginning as indicated by the arrow; I at 0.1 g/L in section 2 on the graph, followed by flushing the cell to remove the I solution, beginning as indicated by the arrow; again introducing aI at 0.1 g/L in section 3 on the graph.
sites, no antigen molecules can react with the adsorbed antibody molecules. On the other hand, many different sites on the antigen molecules can react with different antibody molecules, so that the antigen molecule is much less likely to adsorb so as to hide all possible reaction sites. It should also be observed that the preferential orientation of an adsorbed protein depends both on the surface and on the molecules: the small changes between the antigen and antibody molecules may change the configuration of the adsorbed molecules on the silica and thus the available reaction sites. Our result, with significant differences in the saturation surface concentrations for the two molecules illustrates how sensitive the adsorption process is to small modifications of the adsorbing molecules. This sensitivity to molecule and to surface is certainly one of the reasons it has been difficult to find general laws governing the adsorption process of proteins on solid surfaces. Since the surprisingly large thickness of the antigen/ antibody layer does not seem to be due to aggregation in the bulk or to the presence of larger molecules, some other explanation should be found. One proposition follows: Antigens are adsorbed on the silica surface. The adsorption process involves physical interactions between the molecules and the surface but no covalent bounding. When antibodies react with the adsorbed antigen molecules at the initial stages, an antigen/antibody layer forms. However, over time and due to the fact that the antigen molecules interact with the silica surface only through physical interactions, antibody molecules interacting with the adsorbed layer can also diffuse through it, finally touching the silica surface; during this progression the
Antigen-Antibody Layers
antigen molecules may lose contact with the surface. This process would be similar to the one involved during exchange reactions. However, in the present case the antigen molecule remains attached to the protein layer even if it loses direct contact with the silica surface, while if it was not bound to an antibody, it could simply have left the layer. This “tilting” of the whole antigen/antibody entity would allow the antigen to point toward the solution even if it had already reacted with an antibody. It would thus be available for reacting with other antibody molecules in the solution. Finally, the structure of the layer would be roughly of type antibody/antigen/antibody (see Figure 9b), which would indeed have a thickness of the order of 50-60 nm as observed. The fact that on the average four to five antibody molecules can react with each antigen molecule can be attributed to the polyclonal nature of the antibody solution. This hypothesis is also consistent with the fact that the reorganization of the adsorbed antibody/antigen layer proceeds over a time scale which is of the same order of magnitude as the time scale that characterizes the slow desorption mechanisms.5 One way to test this hypothesis would be to substitute monoclonal antibodies for the polyclonal ones here: in that case, the layer thickness should be no more than twice that of a single antigen or antibody molecule. The quantity of antibodies required is at present prohibitive; miniaturization of the sample cell to allow such studies is under way. IV. Conclusion We have studied the structural evolution of an antigen/ antibody layer when the antibodies react with the antigens which are adsorbed on a silica surface. We find the surprising result that at high antibody concentrations in solution, the layer reaches a maximum thickness which is of the order of 60 nm. This corresponds approximately to 3 times the characteristic size of the proteins. Moreover, we find an antibody/antigen ratio roughly equal to 4:1. Both of these results were found using a particular optical model, that of a homogeneous, isotropic layer, yielding an average thickness and density. The direct structural information is thus limited. However, the overall surface concentrations, and thus the antibody/antigen ratios, are insensitive to the optical model, as is the average overall thickness.25 We can state with reasonable certainty that the maximum thickness of the layer is at least three molecules thick; we cannot, on the basis of the optical signal alone, exclude the possibility of a diffuse protein layer extending further into the solution. This would however imply aggregation of the antigen molecules, which other methods (see section II) appear to exclude. One structure, the only one we have been able to construct, which is compatible with the ensemble of these results would involve a process in which once an antibody molecule (aI) has reacted with an adsorbed antigen protein (I), the complex undergoes a tilting mechanism. In time, the antibody molecules would be in contact with the silica surface and the antigen molecules point toward the solution. New antibody molecules could then again react with the antigen molecules leading finally to a layer of the type antibody/antigen/antibody. Such a mechanism, never to our knowledge observed previously, would be entirely due to the dynamic property of the adsorbed proteins. A similar mechanism has been proposed, among others, for the exchange process.7 The difference is that
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in our case the complex is permanent, due to the specific antibody/antigen interaction, and both molecules remain part of the layer, in contrast to the more general exchange process. The time scale associated with the build-up of the antigen/antibody layer is of the order of 3 days and thus of the same order of magnitude as the characteristic time associated with the slow desorption process of the antigens adsorbed on a silica surface, which is also consistent with such a mechanism. However, experiments which are sensitive to the positions of the two different species separately would be highly desirable to test and explore this hypothesis. Given the similarity of the antigen and antibody molecules, such a method would necessarily involve marking one or both of the types of molecules. Further studies should be performed with other antigen/ antibody systems, and other surfaces, to investigate how this mechanism depends on the particular system. It would also be of interest to perform similar experiments with monoclonal IgG’s. If our postulated mechanism is correct, we should no longer observe the formation of a trilayer. The process should stop after the build-up of a bilayer and the antibody/antigen ratio should be less than 1. Acknowledgment. We thank J.-P. Munch of the Laboratoire d’Ultrasons et Dynamique des Fluides Complexes, Universite´ Louis Pasteur, Strasbourg, for the quasi-static light-scattering experiments, C. Ravanat at the Centre Re´gional de Transfusion Sanguine, Strasbourg, for the electrophoresis tests, the Centre Re´gional de Transfusion Sanguine and J. P. Cazenave for providing us with the proteins, and D. Bedeaux and V. Ball for many fruitful discussions. This work is integrated in the COST action: “Preparation and characterization of model biological interfacessStudy of their interactions with proteins”. Appendix 1 The reflectivity of a homogeneous film, of refractive index n, which consists of two parallel Fresnel interfaces a distance L apart, is well-known;29 it can be calculated from the reflectivity, here for p-polarized light, of Fresnel interfaces between media of refractive index ni and nj
rij )
nj cos θi - ni cos θj nj cos θi + ni cos θj
(A1)
where θi and θj are the angles of incident and refraction, related through the Snell law, ni sin θi ) nj sin θj. The reflectivity of the thin film system is
Rp )
|
r1f + rf2eiδ
|
1 + r1frf2eiδ
(A2)
where the indices 1, f, and 2 refer to the incident medium, the film, and the transmitted medium respectively. δf ) 4πnL cos θf/λ is the phase difference between the field reflected at the two interfaces. LA9602630 (29) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland: Amsterdam, 1989.
