Covalent Binding of Proteins to Acetal-Functionalized Latexes. II. Colloidal Stability and Immunoreactivity

JOURNAL OF COLLOID AND INTERFACE SCIENCE ARTICLE NO.

201, 139–145 (1998)

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Covalent Binding of Proteins to Acetal-Functionalized Latexes. II. Colloidal Stability and Immunoreactivity 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

The present work deals with the study of the colloidal stability and immunoreactivity of acetal-functionalized latex particles covered by different amount of IgG a-CRP protein. This protein has been previously coupled onto the acetal particle surface by covalent binding, and it was possible to obtain latex–protein particles with different degrees of coverage by this protein. The sensitized latex particles were resuspended under several conditions (different pH and ionic strength values), and their colloidal stability was studied by particle size measurements. The latex–protein complexes obtained by covalent binding of the protein show a good colloidal stability at neutral pH and high ionic strength (200 mM), which is a first condition for their application in the immunodiagnostic field. As a final part of this work, the immunoreactivity of several complexes was studied following the changes in the turbidity after the addition of CRP antigen. The immunoreactivity of these complexes depends on their colloidal stability, and treatment with a nonionic surfactant is also important. The surface structure of the latexes has a significant role in the immunological behavior of the complexes because a very high surface charge density can prevent the aggregation of the sensitized particles in the presence of the antigen molecules. The latex–protein complexes obtained by covalent binding show a good immunological response which is not disturbed by the presence of a nonionic surfactant in the reaction medium and is stable with time. q 1998 Academic Press Key Words: protein covalent binding; acetal latex; colloid stability; immunoreactivity.

1. INTRODUCTION

This work is the second in a series of papers studying the interaction of proteins (IgG a-CRP) with acetal polystyrene latexes and the characterization of the particles covered by this protein. The physical adsorption and the covalent coupling onto acetal latexes with different surface charge densities, together with the electrokinetic characterization of the latex–protein complexes obtained, were described in the first of this series (1). However, the complete characterization 1

To whom correspondence should be addressed.

of the latex–protein complexes requires the study of their colloidal stability, as this aspect is very important when looking for its application in the field of clinical diagnostics, where one of the two main requirements is colloidal stability of the latex particles covered by protein. However, there are not many studies on protein chemical adsorption which complete the characterization of the latex–protein samples including their colloidal stability and, if so, the results cannot be generalized to other polymer supports (2, 3) and have to be studied specifically, especially in the case of acetal latex with noncharged groups on the surface. Another requirement for the application of these complexes in clinical diagnostics is the immunoreactivity of the proteins after adsorption or coupling on the particle surface. Although this could be a simple study, again only a scarce number of works complete the immunological application with the immunoreactivity studies (4, 5), and the results cannot be generalized from one to other systems. By covalent binding, the protein can be adsorbed in such a way that the active sites of the protein molecule are directed toward the solution, where they can react with the antigens present in this solution. Thus, the covalent binding can be a method that permit one to obtain immunoreactive particles with several advantages respect to those obtained by physical adsorption (4–9). This covalent coupling prevents the later desorption of the proteins, the active sites are directed toward the solution, and the conformational changes of the protein molecules decrease, improving the specific character of the test. Therefore, we have the possibility of using different complexes with the protein physically or chemically bound, in order to compare their main characteristics. On the other hand, the nonspecific interactions of the immunoreaction between the antigen and the latex–antibody complex are normally removed by the use of a blocking agent of proteinic nature, for example, bovine serum albumin (BSA). However, for a lot of antigen–antibody systems, this type of bloking agent cannot be used because it is possible to find cross-reactions that falsify the result of the immunodiagnostic test. In these cases, the surfactants can be used to

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prevent the nonspecific reactions, but these substances can lead to a desorption of the antibodies physically adsorbed on the latex particles (1, 10, 11). As was seen in the first paper of this series (1), the protein desorption by surfactants can be eliminated with a covalent bond of the antibody to the particle surface. Furthermore, it is also important to establish the influence of the presence of the surfactant on the immunoreactivity of a specific system. In this work, therefore, the colloidal stability and immunoreactivity of the latex–protein complexes obtained by covalent binding of the protein will be studied. To estimate the colloid stability of these complexes their particle sizes will be measured at two pH and different electrolyte concentrations, similar or higher than the physiological ionic strength (150 mM). These sizes have to be compared with the diameter of the bare latexes to observe the aggregation state of the particles covered by protein. The final part of this research is to determine the immunological activity of the IgG that remains coupled on the latex surface. The immunoreactivity of the complexes was measured by optical methods at several experimental conditions for different concentrations of the CRP antigen. In these experiments we have checked the effect of the presence of the surfactant Tween 20 during the redispersion step of the covalent complexes and the particle concentration, comparing the results when the proteins are chemically or physically adsorbed. 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 buffers used were acetate at pH 5, phosphate at pH 7, and borate at pH 8. The salt concentrations were calculated to obtain a final ionic strength of 2 mM. Higher ionic strength values were reached with NaCl. The latex characteristics, protein purification, and preparation of the latex–protein complexes have been previously described (1). Before the experiments of colloid stability and immunoreactivity, all the complexes were cleaned to eliminate the desorbed protein and the excess of surfactant in the solution. The aggregation state of the sensitized latex particles was measured at different pH in order to see the effect of the electric state of the coupled proteins on the colloidal stability of the complexes. We used a static method: the latex particles were first resuspended in 2 mL of buffer, sonicated for 30 s, and then diluted to 0.2 mg mL 01 with the NaCl solutions. These suspensions were kept at room temperature for 12 h, and their aggregation state is determined measuring the particle size of the complexes using a photon correlation spectroscopy (PCS) technique (Malvern 4700C system, Malvern Inst., UK).

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FIG. 1. Particle size of the acetal latexes for several electrolyte concentrations [NaCl]: LK2 latex, pH 5 ( h ); LKN0 latex, pH 5 ( s ) and pH 7 ( 1 ).

The immunoreactivity of the latex particles covered with different amounts of a-CRP–IgG was measured by the changes in the turbidity of the dispersion after several reactions by a spectrophotometer (Spectronic 601, Milton Roy, USA); 0.950 mL of the dispersion containing the latex– protein complex was mixed with 0.050 mL of a solution containing different concentrations of CRP ranging from 0.25 to 40 mg mL 01 and diluted with saline solution (at pH 8) containing 1 mg/mL of BSA (bovine serum albumin) and an ionic strength of 150 mM. The dispersion was stirred and after 5 min the increments in the optical absorbance were measured at a wavelength of 570 nm. The experiment of immunoreactivity was carried out for several complexes, exchanging the BSA protein–saline solution with a 1% concentration of the surfactant Tween 20. 3. RESULTS AND DISCUSSION

3.1. Colloidal Stability of the Latex–IgG Complexes Firstly, the colloidal stability of the acetal latexes was determined by particle size measurements. Figure 1 shows the particle diameter of both acetal latexes under several experimental conditions of pH and ionic strength. The LKN0 latex shows different behavior depending of the pH. This is a consequence of the weak acid character of the surface groups (carboxyl), which begin to protonate at pH 5. Thus, at this pH and an ionic strength of 500 mM the colloidal system is unstable. In this situation the electrostatic repulsion cannot prevent the aggregation of the particles and their diameter increases with time. However, at pH 7, the surface groups are charged and the colloidal stability increases, the particle size remaining constant with time even at an ionic strength of 1 M. The LK2 latex has a colloidal stability independent of the pH in spite of the weak acid character of their surface groups. Figure 1 shows a particle diameter

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FIG. 2. Particle size of LK2–IgG complexes obtained by physical adsorption for different pH and [NaCl]: 1.50 mg/m 2 (black column); 2.37 mg/m 2 (grey column). The numbers in the top of some columns indicate the electrophoretic mobility of the corresponding complexes.

constant at pH 5, from 2 mM to 1 M of ionic strength, and similar results were found for pH 7. Due to its high surface charge density, it is possible the presence of a high amount of oligomers on the surface, ionic and nonionic, as a consequence of the synthesis method (1, 12), which can provide an additional steric contribution to the colloid stability (13– 15). This situation could explain the high colloidal stability found for this type of latex particles. Deeper study into the colloidal stabilization mechanisms of this type of acetal latexes will be the subject of a future work. For latex–protein complexes, a result commonly found is an important decrease in the colloidal stability of the latex particles totally or partially covered by the IgG protein (16, 17). The unstability of the complexes is an important problem from the point of view of the practical application of these systems. Figure 2 shows the particle size at pH 5 and 7 and different ionic strength of two LK2–IgG complexes obtained by physical adsorption. To properly discuss the colloid stability results, at the top of some columns which represent the particle size we show the electrophoretic mobility values of several complexes. As was suggested with the electrophoretic mobility results (1), these complexes were unstable at pH 5, near their isoelectric point (iep), where the mobility values were 02.24 and 01.53 1 10 08 m2 /V s. However, at pH 7 the mobility values were 03.58 and 03.75 1 10 08 m2 /V s, and both complexes maintain their particle size, which means that the particles are dispersed in the solution and that there is no aggregation. This result is very important because we have complexes with IgG on the surface that are stable at neutral pH and high ionic strength. The coverage degree and the special surface characteristics of these acetal latexes seem to be the responsible of this behavior: at pH 7 the carboxyl groups are electrically charged and since the coverage degree (1.50 and 2.37 mg m02 ) is very low (18), the electrostatic repulsion allows the

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expansion of the polymeric surface chains toward the bulk solution. In this way, the approach between particles is not possible, even at high ionic strength when the electrostatic interaction is disminished. The colloidal stability of the LK2–IgG complexes obtained by covalent bound can be evaluated by the particle diameter results shown in Fig. 3. The electrophoretic mobility of some of the complexes is also shown in the top of the columns. These complexes, with an increase in the amount of protein joined (around 3 and 3.8 mg m02 ), present similar behavior in comparison with that previously found for the complexes with the IgG physically adsorbed: they were also stable at pH 7 and unstable at pH 5. The influence of the presence of the surfactant Tween 20 during the redispersion step on the colloid stability of these complexes can be also evaluated in Fig. 3. Those complexes which suffered the action of the surfactant in the redispersion step keep the particle size stable at pH 5 and low ionic strength (2 mM). MartıB n-RodrıB guez (19) have found that the surfactant Tween 20 does not cause an important additional stabilization of polystyrene latex particles by adsorption. As was observed in the previous paper (1), on the basis of the hydrophilic character of the acetal particles and the results of electrophoretic mobility, it is possible to assume an interaction between the surfactant and the protein molecules present on the surface. This interaction would be equivalent to a decrease in the amount of protein bound, and, so, would be responsible for the stabilization at pH 5 and low ionic strength, which is not observed for the complexes without Tween 20. The acetal LKN0 latex presents an important decrease in the surface charge and, therefore, in the amount of surface oligomers. This aspect will be very important in the colloidal stability of the LKN0–IgG complexes. As can be seen in

FIG. 3. Particle size of LK2–IgG complexes obtained by covalent coupling for different pH and [NaCl]: 3.03 mg/m 2 without Tween (light grey column); 3.02 mg/m 2 with Tween (black column); 3.82 mg/m 2 without Tween (white column); 3.81 mg/m 2 with Tween (dark grey column). The numbers in the top of some columns indicate the electrophoretic mobility of the corresponding complexes.

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FIG. 4. Particle size of LKN0–IgG complexes obtained by physical adsorption for different pH and [NaCl]: 2.02 mg/m 2 (black column); 3.78 mg/m 2 (grey column). The numbers in the top of some columns indicate the electrophoretic mobility of the corresponding complexes.

Fig. 4, all the complexes of this latex obtained by physical adsorption were unstables at pH 5, even at low ionic strength (2 mM). When the pH increased up to 7, the complexes with an amount of IgG on the surface equal or higher than 2 mg m02 were also unstable and the particle size rapidly increased when the ionic strength was around the physiological value, 150 mM. In fact, the electrophoretic mobility of these complexes (shown in the top of the columns for some complexes) had low values which indicated their low colloidal stability (1). This result will be checked with the immunoreactivity experiments. At high ionic strength (200 and 500 mM) and pH 5 or 7 there are no mobility measurements due to the fast coagulation of the samples together with the inherent difficulty for measuring at high ionic strength. The particle diameter of the LKN0 latex complexes obtained covalently can be seen in Fig. 5. Also in the top of the columns we show the electrophoretic mobility results of some of the complexes. The behavior of these complexes is different than those obtained by physical adsorption, and a complex with a degree of coverage of 2.74 mg m02 was stable at pH 5 and low ionic strength, even with low electrophoretic mobility values. When the amount of protein increases, 3.93 mg m02 , the particle size also increases with time, which indicates low stability. All the complexes are unstable at pH 5 when the ionic strength is 200 mM. At pH 7 the LKN0 latex complexes were colloidally stable for an ionic strength of 200 mM, although the covered particles redispersed in the absence of Tween 20 show a particular result. The particle sizes of the complexes previously indicated (2.74 and 3.93 mg m02 ) were 320 and 576 nm, respectively, at pH 7 and 200 mM, which are higher than that previously found for the bare LKN0 latex by TEM (190 nm). However, the particle size was constant with time, which means that the aggregates are not growing and the dispersion is formed by dimers or trimers instead of mono-

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mers. At pH 5 and the same ionic strength, however, the particle size rapidly increased with time. Due to the special ‘‘stability’’ of these aggregates (at pH 7), these samples could be used in immunological reactions and, in fact, the results were good, as we will show below. The action of the surfactant Tween 20 is the same than that indicated for the LK2–IgG complexes. Together with the interaction protein/surfactant molecules, for the LKN0– IgG complexes the Tween 20 gives rise to a little desorption of the IgG from the surface, which also contributes to increase the stability of these complexes. For the LK2 latex there are not differences in the stability of the complexes obtained by physics or covalent adsorption. For the LKN0 latex, however, the covalent complexes were more stable than those obtained by physical adsorption. For this latex, with a very low amount of carboxyl groups in comparison with the LK2 latex, the different arrangement of the protein molecules when they are physically or chemically joined will be the reason for the different behavior of these complexes (1). 3.2. Immunoreactivity The last part of this work is to detect the immunoreactivity of the complexes and to compare the response of the complexes with the IgG covalently or physically coupled. In the experiments of immunoreactivity we have also checked the effect of the presence of a surfactant (Tween 20) on the immunological reaction and the particle concentration. In all the experiments the same complex concentration redispersed in solution but without the presence of the CRP antigen was used as a blank. Constant turbidity in these blanks is an indication of the colloidal stability of the complexes at the physiological ionic strength.

FIG. 5. Particle size of latex LKN0–IgG complexes obtained by covalent coupling for different pH and [NaCl]: 2.74 mg/m 2 without Tween (light grey column); 2.56 mg/m 2 with Tween (black column); 3.93 mg/ m 2 without Tween (white column); 3.76 mg/m 2 with Tween (dark grey column). The numbers in the top of some columns indicate the electrophoretic mobility of the corresponding complexes.

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FIG. 6. Optical absorbance change versus the CRP concentration in solution, for complexes with physics adsorption and different particle concentrations (N in part/mL): 2.10 mg IgG/m 2 LK2, N Å 1.4 1 10 10 ( n ); 2.10 mg IgG/m 2 LK2, N Å 6.5 1 10 10 ( h ); 2.37 mg IgG/m 2 LK2, N Å 8 1 10 10 ( s ); 2.02 mg IgG/m 2 LKN0, N Å 4 1 10 10 ( j ); 2.02 mg IgG/ m 2 LKN0, N Å 8 1 10 10 ( l ).

Figure 6 shows the changes in the optical absorbance versus the CRP concentration in solution for several complexes with the IgG physically adsorbed on both latexes. The results obtained for the LK2 latex–protein complexes indicate that an increase in the particle concentration (N) allows an increase in the immunological response. The experimental curves obtained for the LK2 latex complexes show the typical bell curve of the immunoprecipitation reaction, which is proof of the immunological origin of these curves; for the LKN0 latex the results confirm the colloidal unstability previously found for its complexes obtained by physical adsorption. The turbidity of the blank sample changes with time, and the change in the absorbance does not show the typical bell curve, and, except the first point, any concentration of CRP led to the same increment in the optical response. The changes in the optical absorbance of the LK2 latex complexes obtained by covalent bond of the IgG can be observed in Fig. 7. Those complexes that were redispersed in absence of Tween 20 show an immunological response similar to that previously found in Fig. 6 for a similar particle concentration and slightly higher amount of protein on the surface (for the best result: 3.03 against 2.37 mg m02 ). Thus, similar behavior for the covalent and physics adsorbed complexes was found for this latex. Furthermore, the immunological response obtained for the LK2 latex complexes (maximum absorbance around 0.20 units) is low if we compare it with that found for other latex systems (sulfate and sulfonate latexes) previously studied (maximum absorbance of 0.50 units) (17, 20). This result could be a consequence of the surface characteristics of the LK2 latex, with a very high surface charge density. As was previously commented, there are many polycarboxyl chains on its surface which are

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extended toward the solution at pH 8 (pH of the immunoassays). This situation causes an important repulsion between the covered particles and a very high colloidal stability, which could obstruct the access of the antigen molecules to the immunological recognition sites of the antibodies bound to the surface and also prevent aggregation of the particles even in the presence of the antigen. In fact, Kondo et al. (21) showed that the electrostatic particle–particle and particle– antibody interactions are factors that have an important influence on the immunological agglutination. Figure 7 also shows the absorbance changes for the same complexes which have previously suffered the action of the surfactant Tween 20. This previous treatment causes a slight reduction in the amount of protein coupled (1) but cannot explain the important decrease in the immunoreactivity of these complexes that for the experiments with 4 1 10 10 part/mL show a practically negligible change in the optical absorbance. These results could be explained as a consequence of the interaction between the surfactant and the IgG molecules on the particle surface, which could increase the difficulty for the antigen–antibody interaction because the surfactant molecules would screen the specific sites of the coupled antibodies and, therefore, would diminish the immunoresponse of the complexes (22). The immunoreactivity of the LKN0–IgG complexes with different amount of IgG on the surface obtained by covalent way are shown in Fig. 8. The behavior of the complexes redispersed with the presence of Tween 20 is clearly different from those redispersed without this surfactant. In the last case the change in the absorbance does not show the typical bell curve of the immunoprecipitation reaction and gives a plateau when the CRP concentration is higher than 0.1 mg

FIG. 7. Optical absorbance change versus the CRP concentration in solution, for LK2–IgG complexes with covalent coupling and different particle concentrations (N in part/mL): 2.65 mg/m 2 without Tw, N Å 4 1 10 10 ( n ); 2.15 mg/m 2 with Tw, N Å 4 1 10 10 ( m ); 3.03 mg/m 2 without Tw, N Å 4 1 10 10 ( s ); 3.02 mg/m 2 with Tw, N Å 4 1 10 10 ( l ); 3.03 mg/m 2 without Tw, N Å 8 1 10 10 ( h ); 3.02 mg/m 2 with Tw, N Å 8 1 10 10 ( j ).

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FIG. 8. Optical absorbance change versus the CRP concentration in solution, for LKN0–IgG complexes with covalent coupling and particle concentrations, N Å 9 1 10 10 : 2.74 mg/m 2 without Tw ( h ); 2.56 mg/m 2 with Tw ( j ); 3.93 mg/m 2 without Tw ( s ); 3.76 mg/m 2 with Tw ( l ).

mL 01 . This form of the curve could mean a less specific character of the immunological reaction, which seems to saturate the signal changes. This explanation seems to be confirmed by the similar response obtained for two complexes with very different amount of bound antibody (2.74 and 3.93 mg m02 ). The slight colloidal unstability with a pre-aggregation state for these complexes could contribute to this behavior. However, as Fig. 8 shows, the complexes that were redispersed with Tween 20 show curves with the bell form and an increase in the absorbance change when the amount of IgG on the surface increases (2.56 against 3.76 mg m02 ). This result could mean better sensitivity for the antigen–antibody reaction after the treatment with surfactant. A similar conclusion was obtained previously when working with an acetal latex with different surface characteristics (23), namely, lower surface charge density and low number of surface acetal groups. Kapmeyer et al. (6, 24) also indicate that the action of the surfactant is very important to eliminate nonspecific interactions in the immunological reaction. Furthermore, as was previously commented, these complexes are very stable with the same particle size in the immunoreactivity conditions, which can influence the improvement of the immunoreactivity. After comparison of the immunoreactivity results obtained with both acetal latexes (Figs. 7 and 8), a better absorbance change could be displayed for the LKN0 latex complexes than for the LK2 ones. Because both latexes have similar surface concentration of acetal groups, these results seem to be a consequence of the lower number of carboxyl groups on the LKN0 latex. An excessive surface charge density, therefore, disturbs the antigen–antibody reaction and prevents the agglutination of the latex–protein complexes, being less important which latex has the higher number of acetal groups. The presence of surfactants in the reaction medium led to

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a loss of the immunoreactivity for the complexes obtained by physical adsorption because the IgG molecules are desorbed from the latex surface. The covalent bound can eliminate this problem. Thus, as a final test, we checked the effect of the presence of surfactant in the reaction medium on the immunological activity of a covalent complex. Figure 9 shows the result of immunoreactivity when a LKN0–IgG complex was redispersed in the presence of Tween 20 to remove the physically adsorbed protein before the immunological reaction. After the complex was cleaned, the reaction took place with and without the presence of surfactant in the medium. In this case the presence or absence of surfactant does not influence the final result because the effect of the Tween 20 was produced during the first redispersion, when the physically adsorbed protein was removed from the surface particle. Therefore, if we redisperse the complexes in the presence of surfactant, its later addition to the solution does not modify the results about immunoreactivity. The stability of the reactive species with time is another important characteristic of immunodiagnostic test systems. The reactive covered particles must resist long storage periods without modification of their technical characteristics. The covalent complexes, with IgG stably bound to the particle surface, are very interesting from this point of view. The immunological responses of the acetal complexes obtained covalently have been studied after several months, and we have not found differences in their immunoreactivity behavior. Other authors (25, 26) have obtained similar results working with other covalent systems. 4. CONCLUSIONS

The latex–protein complexes obtained by a covalent bound of the protein to the latex surface show good colloidal

FIG. 9. Optical absorbance change versus the amount of CRP in solution, for a complex redispersed in presence of Tween 20 with 2.56 mg IgG/ m 2 and N Å 9 1 10 10 part/mL; without ( j ) and with ( s ) Tween in the reaction medium.

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stability at neutral pH and high ionic strength (200 mM), which is a first condition for their application in the immunodiagnostic field. The immunoreactivity of these complexes depends on their colloidal stability, and treatment with a nonionic surfactant is also important. The surface structure of the latexes has a significant role in the immunological behavior of the complexes because a very high surface charge density can prevent the aggregation of the sensitized particles in the presence of the antigen molecules. The latex– protein complexes obtained by covalent bond show a good immunological response which is not disturbed by the presence of a surfactant in the reaction medium and is stable with time. 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.

REFERENCES 1. Peula, J. M., Hidalgo-Alvarez, R., and de las Nieves, F. J., J. Colloid Interface Sci. 19X, 000–000. 2. Hidalgo-Alvarez, R., and Galisteo-Gonza´lez, F., Heterogen. Chem. Rev. 2, 249 (1995). 3. Norde, W., Cells Matter. 5, 97 (1995). 4. Basinska, T., and Slomkowski, S., Colloid Polym. Sci. 273, 431 (1995). 5. Slomkowski, S., Kowalczyk, M., Trznadel, M., and Kryszewski, M., in ‘‘Hydrogels and Biodegradable Polymers for Bioapplications,’’ p. 173. American Chemical Society, Washington, DC.

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6. Kapmeyer, W. H., Pauly, H. E., and Tuengler, J., Clinical Lab. Anal. 2, 76 (1988). 7. Bale Oniek, M. D., and Warshawsky, A., Colloid Polym. Sci. 269, 139 (1991). 8. Konings, B. L., Pelssers, E. G., Verhoeven, A. J., and Kamps, K. M., Colloids Surf. B: Biointerfaces 1, 69 (1993). 9. Betton, F., Theretz, A., Elaissari, A., and Pichot, C., Colloids Surfaces 1, 97 (1993). 10. Rapoza, R. J., and Horbett, T. A., J. Colloid Interface Sci. 136, 480 (1990). 11. Wahlgren, M. C., and Nygren, H., J. Colloid Interface Sci. 142, 503 (1991). 12. Santos, R. M., and Forcada, J., J. Polym. Sci., Part A. Polym. Chem. 35, 1605 (1997). 13. Napper, D. H., J. Colloid Interface Sci. 32, 106 (1970). 14. Vincent, B., Luckham, P. F., and Waite, F. A., J. Colloid Interface Sci. 73, 508 (1980). 15. Husband, J. C., and Adams, J. M., Colloid Polym. Sci. 270, 1194 (1992). 16. Martin, A., Puig, J., Galisteo, F., Serra, J., and Hidalgo-Alvarez, R., J. Dispersion Sci. Technol. 13(4), 399 (1992). 17. 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). 18. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 19. MartıB n-RodrıB guez, A. Ph.D. Dissertation, University of Granada, 1993. 20. Peula, J. M., Hidalgo-Alvarez, R., and de las Nieves, F. J., J. Biomater. Sci. Polym. Ed. 7, 241 (1995). 21. Kondo, A., Kawano, T., and Higashitani, K., J. Fermentation Bioeng. 73, 435 (1992). 22. Price, C. P., and Newman, D. J., in ‘‘Principles and Practice of Immunoassay.’’ Stockton Press, Stockton, CA, 1991. 23. Peula, J. M., Hidalgo-Alvarez, R., Santos, R., Forcada, J., and de las Nieves, F. J., J. Mater. Sci.: Mater. Med. 6, 779 (1995). 24. Kapmeyer, W. H., Pure Appl. Chem. 63, 1135 (1991). 25. Medcalf, E. A., Newman, D., and Gilboa, D., J. Immunol. Methods 129, 97 (1987). 26. Medcalf, E. A., Newman, D., Gilboa, D., and Price, C., Clin. Chem. 36, 446 (1990).

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