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Covalent Binding of Proteins to Acetal-Functionalized Latexes. I. Physics and Chemical Adsorption and Electrokinetic Characterization
JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.
201, 132–138 (1998)
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Covalent Binding of Proteins to Acetal-Functionalized Latexes. I. Physics and Chemical Adsorption and Electrokinetic Characterization J. M. Peula,* R. Hidalgo-Alvarez,† and F. J. de las Nieves‡ ,1 *Department of Applied Physics II, University of Ma´laga, Ma´laga 29013, Spain; †Biocolloids and Fluids Physics Group, Department of Applied Physics, Faculty of Sciences, University of Granada, Granada 18071, Spain; and ‡Group of Complex Fluids Physics, Department of Applied Physics, University of Almerı´a, Almerı´a 04120, Spain Received July 11, 1997; accepted December 15, 1997
In this work the interaction of an a-CRP IgG protein with functionalized latexes that have acetal groups on their surfaces has been studied. Two acetal latexes with similar amounts of surface acetal groups but different surface charge densities were used. Some experiments on the physical and chemical adsorption of the IgG onto these polystyrene beads have been performed, and several latex– protein complexes with the IgG physically or chemically bound to the surface were obtained by modifying the incubation conditions. In the covalent coupling experiments of the IgG, the physically adsorbed protein was removed by redispersion of the complexes in the presence of a nonionic surfactant (Tween 20). After this treatment the final amount of protein on the latex surface was around 80% of the total protein initially adsorbed. The latex–protein complexes that formed were characterized from the electrokinetic point of view by measuring their electrophoretic mobilities versus the pH, in order to detect any difference between the particles when the protein is physically or chemically coupled. The isoelectric point ( iep ) of the complexes was around pH 4, where they will be unstable because the electrostatic repulsion cannot stabilize the particles. At neutral and basic pH, the electrophoretic mobility values of the latex– protein particles seem to predict a good colloidal stability which is a very important aspect when looking for its application in the field of the clinical diagnostic. The redispersion of the complexes in the presence of Tween 20 modified the electrokinetic behavior of the particles, especially at pH 4 and 5, which seems to indicate an interaction between the surfactant and the protein molecules which could reduce the immunoreactivity of the latex– protein complexes. q 1998 Academic Press Key Words: protein covalent binding; acetal latex; electrokinetic characterization.
1. INTRODUCTION
The use of polymer latex particles in biomedical applications is of great interest, especially in development of new 1
To whom correspondence should be addressed.
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0021-9797/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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immunodiagnostic tests. The recent advances in polymer science have made it possible to produce uniform latexes with desired size and surface characteristics. Thus, it is possible to obtain latex particles with specific functional groups which can covalently bind antibody molecules via amine groups. From the point of view of its immunological application, the covalent bond of proteins to the particle surface prevents the physical desorption of the proteins and retains the native conformation of these biomolecules. Most of the studies of covalent coupling of proteins were developed with carboxylated latexes and require some chemical reaction (1– 3). Recently, several authors (4–7) indicated that the use of aldehyde groups on the surface could simplify the covalent binding of the protein due to the direct reaction between the aldehyde groups of the surface and the amine groups of the protein molecules. However, due to the chemical unstability of the aldehyde groups, they tend to decompose with time, losing their capacity to link the proteins. As suggested by Kapmeyer et al. (8, 9), another possibility is to produce latex particles with acetal groups on the surface. These groups can be transformed to aldehyde groups to produce the covalent coupling of the proteins, by moving the medium to acid pH. In the present work, we have focused our attention on the interaction of an a-CRP (C-reactive protein) IgG protein with functionalized latexes that have acetal groups on their surface. We used two acetal latexes with a similar amounts of surface acetal groups but with different surface charge densities of carboxyl groups (10). As a first step, the physical adsorption of the protein was studied because of the discrepancies found in the adsorption of IgG on several polymer supports (11, 12), which do not include acetal latexes. As a second and main step, some experiments on the chemical bound of the IgG onto these polystyrene beads have been performed under different experimental conditions. Several latex–protein complexes were obtained with the protein physically or chemically bound to the surface. The next step is to characterize the latex–protein com-
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TABLE 1 Latexes Characteristics Latex
Particle diameter (nm)
s0 (mC cm02)
Aldehyde groups (11007 meq cm02)
LK2 LKN0
199 { 7 190 { 5
130 { 8 35.2 { 2.4
1.22 { 0.07 0.94 { 0.05
plexes formed electrokinetically in order to obtain information about the electric state of the new solid–liquid interface and to detect any difference between the particle where the protein is physically or chemically coupled. Thus, we can obtain the isoelectric point (iep) of the complexes, where they will be unstable because the electrostatic repulsion cannot stabilize the particles. These experiments provide a first estimation of the colloidal stability of the latex particles covered by the protein, which is a very important aspect when looking for its application in the field of the clinical diagnostic. Complete characterization of the protein–latex complexes, from the point of view of colloid stability and immunoreactivity, will be discussed in the second part of this work. 2. MATERIALS AND METHODS
All chemicals used in this study were of analytical grade and were used without further purification. Water used in all experiments was double distilled and deionized with a Milli-Q water purification system (Millipore). The functionalized latex particles with acetal groups on its surface were synthetized by means of a core–shell emulsion polymerization in a batch reactor (10). The core was a seed of polystyrene, and the shell was obtained by terpolymerization of styrene, methacrylic acid, and methacryloyl acetaldehyde di(N-methyl)acetal. The methodology, synthesis procedure, and particle size (by TEM) of the latexes have been reported by Santos and Forcada (10), who kindly provided us some samples. The cleaning of the latex samples was carried out using a serum replacement technique. The particle size distribution were obtained by TEM (Technical Services of the University of Granada). The surface charge density, s0 , of the latexes was obtained by conductimetric titration. The content of aldehyde surface groups was determined after acid cleavage of the acetals and titration with hydroxylamine hydrochloride (13). Table 1 shows several characteristics of the acetal latexes used in this study. The proteins IgG, a-CRP, rabbit polyclonal, and m-BSA were kindly donated by Biokit S.A. (Barcelona, Spain). The IgG sample was purified from CRP immunized rabbit serum by ammonium sulfate fractionation followed by anion exchange chromatography. The purified rabbit IgG was stored at 0207C. The purity of the IgG preparation was ascertained
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by immunoelectrophoresis (14). The BSA used in these experiments was monomeric (97 wt% monomers) (14). It was purified from BSA fraction V fatty acid free (PentexMiles) using a 35/600 Bio-Pilot column (Pharmacia). The physical adsorption and the chemical or covalent bond of the proteins were performed at different pHs. For the covalent coupling of the IgG molecules, the acetal latexes were activated by adding HCl to reach pH 2. The amount of adsorbed proteins on the LK2 and LKN0 latexes was determined by using the depletion method described in (14). Furthermore, the amount of IgG covalently bound to the latex surfaces by the aldehyde groups was determined in the presence of the surfactant Tween 20 which is used to remove the protein physically adsorbed (15, 16). In order to be sure that the surfactant was not disturbed during the determination of the amount of protein in solution, we employed two different methods for those measurements. The usual way was to measure the optical absorbance at 280 nm. The results were compared with those obtained by using a protein assay reagent BCA (Pierce, USA). In this second method, we performed a calibration curve in the presence of Tween 20, which will be the conditions for our measurements. The results obtained by both methods were in very good agreement, and, therefore, we can continue by using the simplest optical absorbance. The electrophoretic mobility of the IgG–latex complexes were measured with a Zeta-Sizer IV (Malvern Instruments, UK) by calculating the average of six measurements at a stationary level in a cylindrical cell. In these experiments the latex particle concentration was 0.03 mg mL 01 . The buffers used were acetate at pH 5, phosphate at pH 7, and borate at pH 9. The salt concentrations were calculated to obtain a final ionic strength of 2 mM. Higher ionic strength values were reached with NaCl. 3. RESULTS AND DISCUSSION
The study of the covalent or chemical binding between the particles surface and the antibody molecules is the objective of this work. However, the special surface characteristics of the acetal particles requires a previous study of the physical interaction (adsorption) of the proteins on these particles owing to the discrepancies found about the adsorption of IgG on several polymer supports (11, 12), which do not include acetal latexes. Thus, we can obtain more information about this interaction and to know the significance of the particle surface characteristics on this kind of process. Figures 1 and 2 show the physical adsorption isotherms of the IgG protein on the LK2 and LKN0 latexes, respectively, at pH 5 and 7 and an ionic strength of 2 mM. The physical adsorption occurs because the polymer particles have not been treated at low pH, and, therefore, the surface acetal groups have not been modified to produce the covalent coupling. For the LK2 latex (Fig. 1), the adsorbed protein
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amount at pH 5 is greater than at pH 7 in which the adsorption is of low affinity. This is a consequence of the iep of the IgG molecules, with polyclonal character and a wide range of iep (6.1–8.7), and the important role played by the electrostatic interaction between surface and proteins at low ionic strength (17). At pH 5 the IgG molecules have positive charge, which leads to an important attractive electrostatic interaction between these and the particles surface, with a high negative charge. However, at pH 7 there is a high percentage of negatively charged IgG molecules, and therefore the protein affinity and the adsorbed amount decrease. Similar behavior was found in sulfonate latexes with high surface charge density (14). Other authors (17, 18) using the same protein and studying their adsorption on cationic and anionic latexes showed the significance of the electrostatic interaction in the adsorption process. In Fig. 1 we also show the amount of protein which remain on the latex surface after the redispersion of the latex–protein complexes at pH 8. The desorption of IgG is very significant due to the electrostatic repulsion between the surface and protein, both negatively charged, at this pH. The final amount of IgG remaining on the surface is around 2 mg/m 2 . This amount is lower than that adsorbed on sulfate and sulfonate latexes (14, 19) or other polymer support (11) as a consequence of the hydrophilic character of the LK2 latex which presents a high electrical charge density and a large number of acetal (aldehyde) groups on its surface (20). Finally, Fig. 1 also shows the amount of IgG that remains on the surface of the LK2 latex after the redispersion of the complexes in the presence of a nonionic surfactant, Tween 20. As can be observed, the protein molecules physically adsorbed are removed from the latex surface in a greater part after this treatment, and the amount of physically adsorbed protein that remains is lower than 0.5 mg/m 2 . The same result was
FIG. 1. Adsorption isotherms of a-CRP–IgG onto LK2 latex (I Å 2 mM) and adsorbed amount of protein after the redispersion at pH 8: at pH 5 ( h ) and after desorption ( n ); at pH 7 ( l ) and after desorption ( . ); after desorption in the presence of Tween 20 (*).
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FIG. 2. Adsorption isotherms of a-CRP–IgG onto LKN0 latex ( I Å 2 mM) and adsorbed amount of protein after the redispersion at pH 8: at pH 5 ( h ) and after desorption ( n ); at pH 7 ( l ) and after desorption ( . ).
obtained by Kapmeyer et al. (8, 9) using also acetal latexes. Therefore, the addition of nonionic surfactants in the liquid phase can be a good method to remove the majority of the proteins physically adsorbed after the covalent reaction between the protein molecules and the acetal latex particles. The latex LKN0 has a lower surface charge density and the electrostatic interaction loses intensity. Thus, the adsorbed amount at pH 5 is lower than LK2 latex, while at pH 7 it is higher. The behavior and the affinity of the IgG for the surface at both pH seem to be similar, and both isotherms, pH 5 and 7, are of high affinity. The amount of protein that remains on the surface of this latex after the redispersion of the complexes at pH 8 is almost the same for both pH and around 4 mg/m 2 . There is a lower desorption in comparison with the LK2 latex due to the differences in the surface charge density between both latexes. The maximum adsorption of the IgG protein as a function of the pH is showed in the Fig. 3. The adsorption values found at pH between 5 and 7 confirm our previous results for the adsorption isotherms. At acid pH the higher adsorption corresponds to the more charged latex, LK2, while at neutral and basic pH it is the LKN0 latex that presents a higher amount of adsorbed protein. As was observed previously, this result seems to be a consequence of the electrostatic attraction or repulsion between the proteins and the latex surface. The bell form in these adsorption curves, with a maximum near the iep of the latex–protein complexes, indicates that the hydrophobic forces are predominant in the adsorption process (21–23). However, the electrostatic interaction increases its significance when the surface charge of the polymer particles is increased. Figure 3 also shows the desorption results when the complexes are redispersed at pH 8. There is a great desorption for acid and neutral pH, and now the bell form of the curve disappears and presents a slight continuous decrease of the
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FIG. 3. Maximum adsorption of a-CRP–IgG onto acetal latexes versus the pH (I Å 2 mM) and adsorbed protein after the redispersion at pH 8: IgG–LK2 ( s ) and after desorption ( n ); IgG–LKN0 ( j ) and after desorption ( . ).
adsorbed amount of IgG as the pH increases. In this situation, the particle surface and the protein molecule are negatively charged, and the only force between then is hydrophobic. Thus, the final adsorbed amount of IgG is higher for the less hydrophilic acetal latex, LKN0. From these experiments of physical adsorption we can use several latex–protein complexes with different amounts of IgG on the surface, and their characteristics will be compared with those displayed by the complexes obtained by covalent bound. The IgG is covalently bound to the latex surface by rapid mixture of the latex and the protein at pH 2. After incubation for 30 min at this pH, the dispersion is changed to pH 5 or 7. At pH 2, the acetal groups change to aldehyde groups and it is possible to direct reaction with the amine groups of the protein molecules to form the covalent complex (16). These latex particles, obtained by a core–shell synthesis method, have a high density of carboxyl groups and present in their surface polycarboxyl chains (10), which produce an important electrostatic interaction with the protein molecules (24, 25). To obtain a covalent binding it is necessary that there be close contact between the protein molecules and the aldehyde surface groups (26). If the pH of the medium is higher than the pK of the carboxyl groups, these chains will be charged and extended toward the solution and the covalent bound is not effective due to the electrostatic interaction (16). The chemical reaction, therefore, is developed at the activation pH of the particles latex, pH 2, when the carboxyl groups are protonated and the electrostatic interaction is minimized. The imine bond formed after the reaction is unstable because it has tendency to hydrolyze and give again amine and aldehyde groups. This bond is reduced by the addition of sodium borohydride (NaBH4 ) (4, 8). Figures 4 and 5 show the amount of IgG covalently coupled to both latexes as a function of the protein added into the solution. For each experiment we also show the amount
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FIG. 4. Amount of a-CRP–IgG covalently coupled to the LK2 latex versus the amount of protein added in solution: initially coupled protein (grey column); after redispersion at pH 8 without Tween 20 (black column) and with Tween 20 (white column).
of protein which remains on the surface after the redispersion at pH 8 with or without the presence of a nonionic surfactant (Tween 20). As previously shown, the redispersion at pH 8 produces a significant desorption of the IgG physically adsorbed. In this situation the protein remains adsorbed by means of hydrophobic forces. The redispersion in the presence of a nonionic surfactant in solution is because the surfactant molecules compete with the protein molecules for the surface and can desorb the majority of the protein physically adsorbed (15, 27). Thus, using this method we can remove the protein adsorbed and detect the final amount of protein which is covalently coupled. For all experiments shown in Figs. 4 and 5 the final amount of protein on the polymeric surface is around 80% of the total protein initially adsorbed,
FIG. 5. Amount of a-CRP–IgG covalently coupled to the LKN0 latex versus the amount of protein added in solution: initially coupled protein (grey column); after redispersion at pH 8 without Tween 20 (black column) and with Tween 20 (white column).
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which means that a great percentage of the protein molecules are covalently coupled to the latex surface and cannot be removed. The presence of the surfactant almost does not influence in the desorbed protein amount (specially for the LK2 latex), because the protein that is not chemically bound is removed by means of electrostatic repulsive interactions between the protein and the surface at the redispersion pH (pH 8). Comparing Figs. 4 and 5, it can be observed that similar amount of covalently bound protein is obtained for both latexes. The covalency process in this particles depends of the initial step, which takes place at pH 2, when the electrostatic interaction disappears and the different amount of surface carboxyl groups of the latex has no influence. Once we have several complexes with different amounts of proteins physically or chemically bound on the surface, the next step is to completely characterize the sensitized latexes and we will begin with the electrokinetic point of view. In all cases, to obtain the electrophoretic mobilities of the complexes, the surfactant that does not interact with the surface is removed from the redispersion medium. Figure 6 shows the electrophoretic mobility of several protein–latex complexes obtained for both acetal latexes when the protein is physically adsorbed. For comparison we also show the mobility of the bare latexes. The mobility of the sensitized particles is highly influenced by the presence of protein on the surface at acid pH, where the protein molecules are positively charged. The LK2 latex complexes present an electrophoretic mobility similar to the bare latex at neutral and basic pH. This is a consequence of the very high surface charge density of these particles since the amount of adsorbed protein is not enough to screen it. This behavior allows us to anticipate that these complexes will be colloidally stable at this pH range. In Fig. (6) we can also observe the electrophoretic mobility of the LKN0 latex complexes.
FIG. 6. Electrophoretic mobilities of several latex–protein complexes obtained by physical adsorption: bare LK2 latex ( n ); 1.5 mg IgG/m 2 LK2 ( h ); 2.37 mg IgG/m 2 LK2 ( s ); bare LKN0 latex ( m ); 2.02 mg IgG/m 2 LKN0 ( j ); 3.78 mg IgG/m 2 LKN0 ( l ).
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FIG. 7. Electrophoretic mobilities of several latex (LK2) –protein complexes: bare LK2 latex ( n ); 0.71 mg/m 2 of IgG covalently bound ( h ); 0.61 mg/m 2 of IgG physically adsorbed ( l ); 3.02 mg/m 2 of IgG covalently bound ( s ); 2.37 mg/m 2 of IgG physically adsorbed ( j ).
For these particles, with a lower surface charge density than the LK2 particles, the presence of protein on the surface give rise to an important decrease in the mobility values at neutral and basic pH in comparison with the bare latex, and the isoelectric point (iep) of these complexes is shifted toward higher pH. In this situation the amount of adsorbed protein screens the surface charge of the particles which could have a lower colloidal stability. Figure 7 shows a comparison between the electrophoretic mobilities of the LK2 latex complexes when the IgG molecules are physically or chemically bound. The behavior is similar for the two kinds of complexes when the coverage degree is low and the electrophoretic mobility depends of the surface characteristics of the latex particles. However, differences between both complexes begin to arise when the protein amount on the surface increases, especially at acid pH when the protein molecules have a strong influence on the electrokinetic behavior. Usually, an increase in the amount of protein presents on the surface of the latex particles provokes a displacement of the iep of the complexes toward more raised pH (14, 18, 19). However, a covalent complex with a higher amount of protein than other obtained by physical adsorption have an iep quite lower, which could indicate different surface conformations of the protein according to the form of joint to the particle surface. Thus, for a covalent bound of the protein the capacity to screen the surface charge of the latex particles diminishes when we are near the iep. At neutral or basic pH, where the protein molecules are negatively charged, there are no differences between these complexes and their mobilities are similar to the bare latex, which means a high colloidal stability. The several complexes (physical or chemical adsorption) prepared with the LKN0 latex have a similar behavior, and the differences between both types of samples are also emphasized (results not shown). This could be a consequence
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of the surface characteristics of the polymer particles. When the hydrophilic character of the latex surface decrease, the hydrophobic interactions between the protein molecules and the particles surface increase. With these interactions, because of adsorption process, important conformational rearrangements of the protein molecules take place that do not appear for the chemically joined proteins. Figures 8 and 9 show the electrophoretic mobility of covalent complexes which have been redispersed with or without the presence of the nonionic surfactant Tween 20, during the desorption step. As was indicated previously, this nonionic surfactant interacts with the latex particles by means of hydrophobic forces generating the desorption of the protein molecules physically adsorbed. With hydrophilic particles, as the acetal latexes, the interaction of the surfactant with the surface is low. For this reason the electrophoretic mobility of the LK2 particles is not affected by the presence of the Tween 20 (see Fig. 8). At acid pH, the complexes with the higher amount of protein, around 3.8 mg/m 2 , have mobility values lower than those around 3 mg/m 2 . For neutral or basic pH, the mobility values depend on the very high surface charge density of these particles and the presence of the surfactant does not affect the behavior of the complexes. However, at pH 4, the carboxyl groups begin to protonate and the protein molecules are positively charged. In this situation the protein screens the surface charge and even change the mobility to positive values for the complex with 3.8 mg/m 2 . When the Tween is present in the redispersion step, the protein molecules joined to the surface seem to lose capacity to screen the surface charge, perhaps as a consequence of the possible interaction of the Tween 20 with the protein molecules (27–29). An additional desorption of protein molecules is not assumed because the desorption process was carried out before the electrophoretic mobility measurements.
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FIG. 9. Electrophoretic mobilities of several latex (LKN0) –protein complexes obtained by covalently bound: bare latex ( n ); 2.74 mg IgG/ m 2 without Tw ( h ); 2.56 mg IgG/m 2 with Tw ( j ); 3.93 mg IgG/m 2 without Tw ( s ); 3.76 mg IgG/m 2 with Tw ( l ).
The results obtained for the electrophoretic mobility of the LKN0 complexes (Fig. 9) seem to confirm the previous idea about the interaction of the surfactant and the protein. The electric state of these complexes is influenced by the protein molecules due to the important reduction in the surface charge of these latex particles. Thus, the action of the surfactant is more marked than in the previous latex and the complexes redispersed in the presence of Tween present mobility values higher than those redispersed without Tween. A significant result is obtained for a complex with 3.76 mg/m 2 which shows an electrophoretic mobility higher than other with 2.74 mg/m 2 redispersed without the surfactant. The Tween 20–protein interaction could modify the biological activity of the protein molecules and influence the immunological response of the complexes. Deeper study of the colloidal stability and the immunoreactivity of the acetal latex–protein complexes is the subject of the accompanying paper. But, in any case, the important notice is the electrophoretic mobility values of the samples which seem to indicate a good colloidal stability of all the complexes at neutral or basic charge. 4. CONCLUSIONS
FIG. 8. Electrophoretic mobilities of several latex (LK2) –protein complexes obtained by covalently bound: bare latex ( n ); bare latex with Tw ( 1 ); 3.02 mg IgG/m 2 without Tw ( h ); 3.03 mg IgG/m 2 with Tw ( j ); 3.82 mg IgG/m 2 without Tw ( s ); 3.81 mg IgG/m 2 with Tw ( l ).
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The use of acetal latex has permitted the covalent adsorption of a significant amount of protein molecules. This reaction takes place simply by changing the pH of the suspension to pH 2, without a preactivation step. The surface characteristics of the latex particles play an important role in the electrokinetic behavior of the complexes. When the a-CRP– IgG is covalently bound, the complexes show iep below pH 5 and high electrophoretic mobility values at neutral or basic pH. This situation indicates that these complexes will be colloidally stable. The presence of the surfactant Tween 20,
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to ensure formation of the covalent bond, could modify the immunological activity of the complexes due to the interaction between the surfactant and the protein molecules. ACKNOWLEDGMENTS This work is supported by the Comisio´n Interministerial de Ciencia y TecnologıB a (CICYT), under project MAT 96-1035-C03-03. The authors thank Dr. R. Santos and Dr. J. Forcada, from the University of the Basque Country, for kindly supplying the latex samples.
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11. Hidalgo-Alvarez, R., and Galisteo-Gonza´lez, F., Heterogen. Chem. Rev. 2, 249 (1995). 12. Norde, W., Cells Mater. 5, 97 (1995). 13. Yan, Ch., Zhang, X., Sun, Z., Kitano, H., and Ise, N., J. Appl. Polym. Sci. 40, 89 (1990). 14. Peula, J. M., Hidalgo-Alvarez, R., and de las Nieves, F. J., J. Biomater. Sci. Polym. Ed. 7, 231 (1995). 15. Lichtenbelt, J. W., Heuvelsland, W. J., Oldenzeel, M. E., and Zsom, R. L., Colloids Surf. B: Biointerfaces 1, 75 (1993). 16. Peula, J. M., Hidalgo-Alvarez, R., Santos, R., Forcada, J., and de las Nieves, F. J., J. Mater. Sci.: Mater. Med. 6, 779 (1995). 17. Martin, A., Puig, J., Galisteo, F., Serra, J., and Hidalgo-Alvarez, R., J. Dispersion Sci. Technol. 13(4), 399 (1992). 18. Galisteo, F., Puig, J., Martin, A., Serra, J., and Hidalgo-Alvarez, R., Colloids Surf. B: Biointerfaces 2, 435 (1994). 19. Peula, J. M., Puig, J., Serra, J., de las Nieves, F. J., and Hidalgo-Alvarez, R., Colloids Surf. A: Physicochem. Eng. Aspects 92, 127 (1994). 20. Bastos, D., Santos, R., Forcada, J., Hidalgo-Alvarez, R., and de las Nieves, F. J., Colloids Surf. A: Physicochem. Eng. Aspects 92, 137 (1994). 21. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 22. Bagchi, P., and Birnbaum, S. N., J. Colloid Interface Sci. 83, 460 (1981). 23. Elgersma, A. V., Zsom, R. L. J., Norde, W., and Lyklema, J., Colloids Surf. 54, 145 (1991). 24. Suzawa, T., Shirahama, H., and Matsumura, Y., Colloids Surf. 56, 293 (1982). 25. Lee, S. H., and Ruckenstein, E., J. Colloid Interface Sci. 125, 365 (1988). 26. Konings, B. L., Pelssers, E. G., Verhoeven, A. J., and Kamps, K. M., Colloids Surf. B: Biointerfaces 1, 69 (1993). 27. Gou, X. H., Zhao, N. M., Chen, S. H., and Teixeira, J., Biopolymers 29, 335 (1990). 28. Coke, M., Wilde, P. J., Rusell, E. J., and Clark, D. C., J. Colloid Interface Sci. 138, 489 (1990). 29. Courthaudon, J. L., Dickinson, E., and Matsumura, Y., Colloid Surf. 56, 293 (1991).
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Covalent Binding of Proteins to Acetal-Functionalized Latexes. I. Physics and Chemical Adsorption and Electrokinetic Characterization J. M. Peula,* R. Hidalgo-Alvarez,† and F. J. de las Nieves‡ ,1 *Department of Applied Physics II, University of Ma´laga, Ma´laga 29013, Spain; †Biocolloids and Fluids Physics Group, Department of Applied Physics, Faculty of Sciences, University of Granada, Granada 18071, Spain; and ‡Group of Complex Fluids Physics, Department of Applied Physics, University of Almerı´a, Almerı´a 04120, Spain Received July 11, 1997; accepted December 15, 1997
In this work the interaction of an a-CRP IgG protein with functionalized latexes that have acetal groups on their surfaces has been studied. Two acetal latexes with similar amounts of surface acetal groups but different surface charge densities were used. Some experiments on the physical and chemical adsorption of the IgG onto these polystyrene beads have been performed, and several latex– protein complexes with the IgG physically or chemically bound to the surface were obtained by modifying the incubation conditions. In the covalent coupling experiments of the IgG, the physically adsorbed protein was removed by redispersion of the complexes in the presence of a nonionic surfactant (Tween 20). After this treatment the final amount of protein on the latex surface was around 80% of the total protein initially adsorbed. The latex–protein complexes that formed were characterized from the electrokinetic point of view by measuring their electrophoretic mobilities versus the pH, in order to detect any difference between the particles when the protein is physically or chemically coupled. The isoelectric point ( iep ) of the complexes was around pH 4, where they will be unstable because the electrostatic repulsion cannot stabilize the particles. At neutral and basic pH, the electrophoretic mobility values of the latex– protein particles seem to predict a good colloidal stability which is a very important aspect when looking for its application in the field of the clinical diagnostic. The redispersion of the complexes in the presence of Tween 20 modified the electrokinetic behavior of the particles, especially at pH 4 and 5, which seems to indicate an interaction between the surfactant and the protein molecules which could reduce the immunoreactivity of the latex– protein complexes. q 1998 Academic Press Key Words: protein covalent binding; acetal latex; electrokinetic characterization.
1. INTRODUCTION
The use of polymer latex particles in biomedical applications is of great interest, especially in development of new 1
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immunodiagnostic tests. The recent advances in polymer science have made it possible to produce uniform latexes with desired size and surface characteristics. Thus, it is possible to obtain latex particles with specific functional groups which can covalently bind antibody molecules via amine groups. From the point of view of its immunological application, the covalent bond of proteins to the particle surface prevents the physical desorption of the proteins and retains the native conformation of these biomolecules. Most of the studies of covalent coupling of proteins were developed with carboxylated latexes and require some chemical reaction (1– 3). Recently, several authors (4–7) indicated that the use of aldehyde groups on the surface could simplify the covalent binding of the protein due to the direct reaction between the aldehyde groups of the surface and the amine groups of the protein molecules. However, due to the chemical unstability of the aldehyde groups, they tend to decompose with time, losing their capacity to link the proteins. As suggested by Kapmeyer et al. (8, 9), another possibility is to produce latex particles with acetal groups on the surface. These groups can be transformed to aldehyde groups to produce the covalent coupling of the proteins, by moving the medium to acid pH. In the present work, we have focused our attention on the interaction of an a-CRP (C-reactive protein) IgG protein with functionalized latexes that have acetal groups on their surface. We used two acetal latexes with a similar amounts of surface acetal groups but with different surface charge densities of carboxyl groups (10). As a first step, the physical adsorption of the protein was studied because of the discrepancies found in the adsorption of IgG on several polymer supports (11, 12), which do not include acetal latexes. As a second and main step, some experiments on the chemical bound of the IgG onto these polystyrene beads have been performed under different experimental conditions. Several latex–protein complexes were obtained with the protein physically or chemically bound to the surface. The next step is to characterize the latex–protein com-
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TABLE 1 Latexes Characteristics Latex
Particle diameter (nm)
s0 (mC cm02)
Aldehyde groups (11007 meq cm02)
LK2 LKN0
199 { 7 190 { 5
130 { 8 35.2 { 2.4
1.22 { 0.07 0.94 { 0.05
plexes formed electrokinetically in order to obtain information about the electric state of the new solid–liquid interface and to detect any difference between the particle where the protein is physically or chemically coupled. Thus, we can obtain the isoelectric point (iep) of the complexes, where they will be unstable because the electrostatic repulsion cannot stabilize the particles. These experiments provide a first estimation of the colloidal stability of the latex particles covered by the protein, which is a very important aspect when looking for its application in the field of the clinical diagnostic. Complete characterization of the protein–latex complexes, from the point of view of colloid stability and immunoreactivity, will be discussed in the second part of this work. 2. MATERIALS AND METHODS
All chemicals used in this study were of analytical grade and were used without further purification. Water used in all experiments was double distilled and deionized with a Milli-Q water purification system (Millipore). The functionalized latex particles with acetal groups on its surface were synthetized by means of a core–shell emulsion polymerization in a batch reactor (10). The core was a seed of polystyrene, and the shell was obtained by terpolymerization of styrene, methacrylic acid, and methacryloyl acetaldehyde di(N-methyl)acetal. The methodology, synthesis procedure, and particle size (by TEM) of the latexes have been reported by Santos and Forcada (10), who kindly provided us some samples. The cleaning of the latex samples was carried out using a serum replacement technique. The particle size distribution were obtained by TEM (Technical Services of the University of Granada). The surface charge density, s0 , of the latexes was obtained by conductimetric titration. The content of aldehyde surface groups was determined after acid cleavage of the acetals and titration with hydroxylamine hydrochloride (13). Table 1 shows several characteristics of the acetal latexes used in this study. The proteins IgG, a-CRP, rabbit polyclonal, and m-BSA were kindly donated by Biokit S.A. (Barcelona, Spain). The IgG sample was purified from CRP immunized rabbit serum by ammonium sulfate fractionation followed by anion exchange chromatography. The purified rabbit IgG was stored at 0207C. The purity of the IgG preparation was ascertained
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by immunoelectrophoresis (14). The BSA used in these experiments was monomeric (97 wt% monomers) (14). It was purified from BSA fraction V fatty acid free (PentexMiles) using a 35/600 Bio-Pilot column (Pharmacia). The physical adsorption and the chemical or covalent bond of the proteins were performed at different pHs. For the covalent coupling of the IgG molecules, the acetal latexes were activated by adding HCl to reach pH 2. The amount of adsorbed proteins on the LK2 and LKN0 latexes was determined by using the depletion method described in (14). Furthermore, the amount of IgG covalently bound to the latex surfaces by the aldehyde groups was determined in the presence of the surfactant Tween 20 which is used to remove the protein physically adsorbed (15, 16). In order to be sure that the surfactant was not disturbed during the determination of the amount of protein in solution, we employed two different methods for those measurements. The usual way was to measure the optical absorbance at 280 nm. The results were compared with those obtained by using a protein assay reagent BCA (Pierce, USA). In this second method, we performed a calibration curve in the presence of Tween 20, which will be the conditions for our measurements. The results obtained by both methods were in very good agreement, and, therefore, we can continue by using the simplest optical absorbance. The electrophoretic mobility of the IgG–latex complexes were measured with a Zeta-Sizer IV (Malvern Instruments, UK) by calculating the average of six measurements at a stationary level in a cylindrical cell. In these experiments the latex particle concentration was 0.03 mg mL 01 . The buffers used were acetate at pH 5, phosphate at pH 7, and borate at pH 9. The salt concentrations were calculated to obtain a final ionic strength of 2 mM. Higher ionic strength values were reached with NaCl. 3. RESULTS AND DISCUSSION
The study of the covalent or chemical binding between the particles surface and the antibody molecules is the objective of this work. However, the special surface characteristics of the acetal particles requires a previous study of the physical interaction (adsorption) of the proteins on these particles owing to the discrepancies found about the adsorption of IgG on several polymer supports (11, 12), which do not include acetal latexes. Thus, we can obtain more information about this interaction and to know the significance of the particle surface characteristics on this kind of process. Figures 1 and 2 show the physical adsorption isotherms of the IgG protein on the LK2 and LKN0 latexes, respectively, at pH 5 and 7 and an ionic strength of 2 mM. The physical adsorption occurs because the polymer particles have not been treated at low pH, and, therefore, the surface acetal groups have not been modified to produce the covalent coupling. For the LK2 latex (Fig. 1), the adsorbed protein
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amount at pH 5 is greater than at pH 7 in which the adsorption is of low affinity. This is a consequence of the iep of the IgG molecules, with polyclonal character and a wide range of iep (6.1–8.7), and the important role played by the electrostatic interaction between surface and proteins at low ionic strength (17). At pH 5 the IgG molecules have positive charge, which leads to an important attractive electrostatic interaction between these and the particles surface, with a high negative charge. However, at pH 7 there is a high percentage of negatively charged IgG molecules, and therefore the protein affinity and the adsorbed amount decrease. Similar behavior was found in sulfonate latexes with high surface charge density (14). Other authors (17, 18) using the same protein and studying their adsorption on cationic and anionic latexes showed the significance of the electrostatic interaction in the adsorption process. In Fig. 1 we also show the amount of protein which remain on the latex surface after the redispersion of the latex–protein complexes at pH 8. The desorption of IgG is very significant due to the electrostatic repulsion between the surface and protein, both negatively charged, at this pH. The final amount of IgG remaining on the surface is around 2 mg/m 2 . This amount is lower than that adsorbed on sulfate and sulfonate latexes (14, 19) or other polymer support (11) as a consequence of the hydrophilic character of the LK2 latex which presents a high electrical charge density and a large number of acetal (aldehyde) groups on its surface (20). Finally, Fig. 1 also shows the amount of IgG that remains on the surface of the LK2 latex after the redispersion of the complexes in the presence of a nonionic surfactant, Tween 20. As can be observed, the protein molecules physically adsorbed are removed from the latex surface in a greater part after this treatment, and the amount of physically adsorbed protein that remains is lower than 0.5 mg/m 2 . The same result was
FIG. 1. Adsorption isotherms of a-CRP–IgG onto LK2 latex (I Å 2 mM) and adsorbed amount of protein after the redispersion at pH 8: at pH 5 ( h ) and after desorption ( n ); at pH 7 ( l ) and after desorption ( . ); after desorption in the presence of Tween 20 (*).
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FIG. 2. Adsorption isotherms of a-CRP–IgG onto LKN0 latex ( I Å 2 mM) and adsorbed amount of protein after the redispersion at pH 8: at pH 5 ( h ) and after desorption ( n ); at pH 7 ( l ) and after desorption ( . ).
obtained by Kapmeyer et al. (8, 9) using also acetal latexes. Therefore, the addition of nonionic surfactants in the liquid phase can be a good method to remove the majority of the proteins physically adsorbed after the covalent reaction between the protein molecules and the acetal latex particles. The latex LKN0 has a lower surface charge density and the electrostatic interaction loses intensity. Thus, the adsorbed amount at pH 5 is lower than LK2 latex, while at pH 7 it is higher. The behavior and the affinity of the IgG for the surface at both pH seem to be similar, and both isotherms, pH 5 and 7, are of high affinity. The amount of protein that remains on the surface of this latex after the redispersion of the complexes at pH 8 is almost the same for both pH and around 4 mg/m 2 . There is a lower desorption in comparison with the LK2 latex due to the differences in the surface charge density between both latexes. The maximum adsorption of the IgG protein as a function of the pH is showed in the Fig. 3. The adsorption values found at pH between 5 and 7 confirm our previous results for the adsorption isotherms. At acid pH the higher adsorption corresponds to the more charged latex, LK2, while at neutral and basic pH it is the LKN0 latex that presents a higher amount of adsorbed protein. As was observed previously, this result seems to be a consequence of the electrostatic attraction or repulsion between the proteins and the latex surface. The bell form in these adsorption curves, with a maximum near the iep of the latex–protein complexes, indicates that the hydrophobic forces are predominant in the adsorption process (21–23). However, the electrostatic interaction increases its significance when the surface charge of the polymer particles is increased. Figure 3 also shows the desorption results when the complexes are redispersed at pH 8. There is a great desorption for acid and neutral pH, and now the bell form of the curve disappears and presents a slight continuous decrease of the
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FIG. 3. Maximum adsorption of a-CRP–IgG onto acetal latexes versus the pH (I Å 2 mM) and adsorbed protein after the redispersion at pH 8: IgG–LK2 ( s ) and after desorption ( n ); IgG–LKN0 ( j ) and after desorption ( . ).
adsorbed amount of IgG as the pH increases. In this situation, the particle surface and the protein molecule are negatively charged, and the only force between then is hydrophobic. Thus, the final adsorbed amount of IgG is higher for the less hydrophilic acetal latex, LKN0. From these experiments of physical adsorption we can use several latex–protein complexes with different amounts of IgG on the surface, and their characteristics will be compared with those displayed by the complexes obtained by covalent bound. The IgG is covalently bound to the latex surface by rapid mixture of the latex and the protein at pH 2. After incubation for 30 min at this pH, the dispersion is changed to pH 5 or 7. At pH 2, the acetal groups change to aldehyde groups and it is possible to direct reaction with the amine groups of the protein molecules to form the covalent complex (16). These latex particles, obtained by a core–shell synthesis method, have a high density of carboxyl groups and present in their surface polycarboxyl chains (10), which produce an important electrostatic interaction with the protein molecules (24, 25). To obtain a covalent binding it is necessary that there be close contact between the protein molecules and the aldehyde surface groups (26). If the pH of the medium is higher than the pK of the carboxyl groups, these chains will be charged and extended toward the solution and the covalent bound is not effective due to the electrostatic interaction (16). The chemical reaction, therefore, is developed at the activation pH of the particles latex, pH 2, when the carboxyl groups are protonated and the electrostatic interaction is minimized. The imine bond formed after the reaction is unstable because it has tendency to hydrolyze and give again amine and aldehyde groups. This bond is reduced by the addition of sodium borohydride (NaBH4 ) (4, 8). Figures 4 and 5 show the amount of IgG covalently coupled to both latexes as a function of the protein added into the solution. For each experiment we also show the amount
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FIG. 4. Amount of a-CRP–IgG covalently coupled to the LK2 latex versus the amount of protein added in solution: initially coupled protein (grey column); after redispersion at pH 8 without Tween 20 (black column) and with Tween 20 (white column).
of protein which remains on the surface after the redispersion at pH 8 with or without the presence of a nonionic surfactant (Tween 20). As previously shown, the redispersion at pH 8 produces a significant desorption of the IgG physically adsorbed. In this situation the protein remains adsorbed by means of hydrophobic forces. The redispersion in the presence of a nonionic surfactant in solution is because the surfactant molecules compete with the protein molecules for the surface and can desorb the majority of the protein physically adsorbed (15, 27). Thus, using this method we can remove the protein adsorbed and detect the final amount of protein which is covalently coupled. For all experiments shown in Figs. 4 and 5 the final amount of protein on the polymeric surface is around 80% of the total protein initially adsorbed,
FIG. 5. Amount of a-CRP–IgG covalently coupled to the LKN0 latex versus the amount of protein added in solution: initially coupled protein (grey column); after redispersion at pH 8 without Tween 20 (black column) and with Tween 20 (white column).
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which means that a great percentage of the protein molecules are covalently coupled to the latex surface and cannot be removed. The presence of the surfactant almost does not influence in the desorbed protein amount (specially for the LK2 latex), because the protein that is not chemically bound is removed by means of electrostatic repulsive interactions between the protein and the surface at the redispersion pH (pH 8). Comparing Figs. 4 and 5, it can be observed that similar amount of covalently bound protein is obtained for both latexes. The covalency process in this particles depends of the initial step, which takes place at pH 2, when the electrostatic interaction disappears and the different amount of surface carboxyl groups of the latex has no influence. Once we have several complexes with different amounts of proteins physically or chemically bound on the surface, the next step is to completely characterize the sensitized latexes and we will begin with the electrokinetic point of view. In all cases, to obtain the electrophoretic mobilities of the complexes, the surfactant that does not interact with the surface is removed from the redispersion medium. Figure 6 shows the electrophoretic mobility of several protein–latex complexes obtained for both acetal latexes when the protein is physically adsorbed. For comparison we also show the mobility of the bare latexes. The mobility of the sensitized particles is highly influenced by the presence of protein on the surface at acid pH, where the protein molecules are positively charged. The LK2 latex complexes present an electrophoretic mobility similar to the bare latex at neutral and basic pH. This is a consequence of the very high surface charge density of these particles since the amount of adsorbed protein is not enough to screen it. This behavior allows us to anticipate that these complexes will be colloidally stable at this pH range. In Fig. (6) we can also observe the electrophoretic mobility of the LKN0 latex complexes.
FIG. 6. Electrophoretic mobilities of several latex–protein complexes obtained by physical adsorption: bare LK2 latex ( n ); 1.5 mg IgG/m 2 LK2 ( h ); 2.37 mg IgG/m 2 LK2 ( s ); bare LKN0 latex ( m ); 2.02 mg IgG/m 2 LKN0 ( j ); 3.78 mg IgG/m 2 LKN0 ( l ).
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FIG. 7. Electrophoretic mobilities of several latex (LK2) –protein complexes: bare LK2 latex ( n ); 0.71 mg/m 2 of IgG covalently bound ( h ); 0.61 mg/m 2 of IgG physically adsorbed ( l ); 3.02 mg/m 2 of IgG covalently bound ( s ); 2.37 mg/m 2 of IgG physically adsorbed ( j ).
For these particles, with a lower surface charge density than the LK2 particles, the presence of protein on the surface give rise to an important decrease in the mobility values at neutral and basic pH in comparison with the bare latex, and the isoelectric point (iep) of these complexes is shifted toward higher pH. In this situation the amount of adsorbed protein screens the surface charge of the particles which could have a lower colloidal stability. Figure 7 shows a comparison between the electrophoretic mobilities of the LK2 latex complexes when the IgG molecules are physically or chemically bound. The behavior is similar for the two kinds of complexes when the coverage degree is low and the electrophoretic mobility depends of the surface characteristics of the latex particles. However, differences between both complexes begin to arise when the protein amount on the surface increases, especially at acid pH when the protein molecules have a strong influence on the electrokinetic behavior. Usually, an increase in the amount of protein presents on the surface of the latex particles provokes a displacement of the iep of the complexes toward more raised pH (14, 18, 19). However, a covalent complex with a higher amount of protein than other obtained by physical adsorption have an iep quite lower, which could indicate different surface conformations of the protein according to the form of joint to the particle surface. Thus, for a covalent bound of the protein the capacity to screen the surface charge of the latex particles diminishes when we are near the iep. At neutral or basic pH, where the protein molecules are negatively charged, there are no differences between these complexes and their mobilities are similar to the bare latex, which means a high colloidal stability. The several complexes (physical or chemical adsorption) prepared with the LKN0 latex have a similar behavior, and the differences between both types of samples are also emphasized (results not shown). This could be a consequence
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of the surface characteristics of the polymer particles. When the hydrophilic character of the latex surface decrease, the hydrophobic interactions between the protein molecules and the particles surface increase. With these interactions, because of adsorption process, important conformational rearrangements of the protein molecules take place that do not appear for the chemically joined proteins. Figures 8 and 9 show the electrophoretic mobility of covalent complexes which have been redispersed with or without the presence of the nonionic surfactant Tween 20, during the desorption step. As was indicated previously, this nonionic surfactant interacts with the latex particles by means of hydrophobic forces generating the desorption of the protein molecules physically adsorbed. With hydrophilic particles, as the acetal latexes, the interaction of the surfactant with the surface is low. For this reason the electrophoretic mobility of the LK2 particles is not affected by the presence of the Tween 20 (see Fig. 8). At acid pH, the complexes with the higher amount of protein, around 3.8 mg/m 2 , have mobility values lower than those around 3 mg/m 2 . For neutral or basic pH, the mobility values depend on the very high surface charge density of these particles and the presence of the surfactant does not affect the behavior of the complexes. However, at pH 4, the carboxyl groups begin to protonate and the protein molecules are positively charged. In this situation the protein screens the surface charge and even change the mobility to positive values for the complex with 3.8 mg/m 2 . When the Tween is present in the redispersion step, the protein molecules joined to the surface seem to lose capacity to screen the surface charge, perhaps as a consequence of the possible interaction of the Tween 20 with the protein molecules (27–29). An additional desorption of protein molecules is not assumed because the desorption process was carried out before the electrophoretic mobility measurements.
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FIG. 9. Electrophoretic mobilities of several latex (LKN0) –protein complexes obtained by covalently bound: bare latex ( n ); 2.74 mg IgG/ m 2 without Tw ( h ); 2.56 mg IgG/m 2 with Tw ( j ); 3.93 mg IgG/m 2 without Tw ( s ); 3.76 mg IgG/m 2 with Tw ( l ).
The results obtained for the electrophoretic mobility of the LKN0 complexes (Fig. 9) seem to confirm the previous idea about the interaction of the surfactant and the protein. The electric state of these complexes is influenced by the protein molecules due to the important reduction in the surface charge of these latex particles. Thus, the action of the surfactant is more marked than in the previous latex and the complexes redispersed in the presence of Tween present mobility values higher than those redispersed without Tween. A significant result is obtained for a complex with 3.76 mg/m 2 which shows an electrophoretic mobility higher than other with 2.74 mg/m 2 redispersed without the surfactant. The Tween 20–protein interaction could modify the biological activity of the protein molecules and influence the immunological response of the complexes. Deeper study of the colloidal stability and the immunoreactivity of the acetal latex–protein complexes is the subject of the accompanying paper. But, in any case, the important notice is the electrophoretic mobility values of the samples which seem to indicate a good colloidal stability of all the complexes at neutral or basic charge. 4. CONCLUSIONS
FIG. 8. Electrophoretic mobilities of several latex (LK2) –protein complexes obtained by covalently bound: bare latex ( n ); bare latex with Tw ( 1 ); 3.02 mg IgG/m 2 without Tw ( h ); 3.03 mg IgG/m 2 with Tw ( j ); 3.82 mg IgG/m 2 without Tw ( s ); 3.81 mg IgG/m 2 with Tw ( l ).
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The use of acetal latex has permitted the covalent adsorption of a significant amount of protein molecules. This reaction takes place simply by changing the pH of the suspension to pH 2, without a preactivation step. The surface characteristics of the latex particles play an important role in the electrokinetic behavior of the complexes. When the a-CRP– IgG is covalently bound, the complexes show iep below pH 5 and high electrophoretic mobility values at neutral or basic pH. This situation indicates that these complexes will be colloidally stable. The presence of the surfactant Tween 20,
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to ensure formation of the covalent bond, could modify the immunological activity of the complexes due to the interaction between the surfactant and the protein molecules. ACKNOWLEDGMENTS This work is supported by the Comisio´n Interministerial de Ciencia y TecnologıB a (CICYT), under project MAT 96-1035-C03-03. The authors thank Dr. R. Santos and Dr. J. Forcada, from the University of the Basque Country, for kindly supplying the latex samples.
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