Langmuir 1996, 12, 3211-3220
3211
F(ab′)2-Coated Polymer Carriers: Electrokinetic Behavior and Colloidal Stability J. L. Ortega Vinuesa, M. J. Ga´lvez Ruiz, and R. Hidalgo-A Ä lvarez* Biocolloid and Fluid Physics Group, Department of Applied Physics, Faculty of Sciences, Campus Fuentenueva, University of Granada, Granada 18071, Spain Received December 14, 1995. In Final Form: March 18, 1996X The main goal of the present work is to gain insight into the mechanisms and forces that govern the adsorption of F(ab′)2 fragments onto different polymer surfaces. We have employed F(ab′)2 fragments obtained by pepsin digestion of rabbit polyclonal IgG. Colloidal polystyrene particles with different superficial characteristics have been used as polymer carriers. These latex particles differ in their hydrophobic/ hydrophilic character, surface charge density, and the nature of the ionic groups located at their solidwater interface. We have checked that the most important driving force during the F(ab′)2 adsorption is the hydrophobic one. However, when adsorption isotherms are performed in media of low ionic strength, the electrostatic interaction plays an important role. First, adsorption experiments carried out, followed by both desorption studies and electrokinetic characterization of the F(ab′)2-latex complexes. The results of the electrokinetic characterization are in good agreement with those obtained from the stability studies. The research presented in this paper throws light on interesting aspects important when developing immunoassay reagents.
1. Introduction The interaction between antibodies or antigens and polymer colloids is of considerable importance when developing particle-enhanced immunoassays. The use of polyclonal IgG molecules in latex immunoassays (LIAs) can give rise to immunolatexes with a very low colloidal stability.1 Consequently, the sensitized particles can coagulate in the absence of the conjugated antigen, due to the ionic strength of the reaction medium (usually 150170 mM). Also, the Fc moieties of IgG molecules are involved in nonspecific interactions with substances that do not operate as antigens, i.e. rheumatoid factors,2 which leads to a nonspecific agglutination of the colloidal system, giving rise to a false positive diagnostic. This is why there is a growing interest in adsorption studies using IgG fragments instead of the whole molecule. Recently, it has been demonstrated that the use of the F(ab′)2 antibody fragment is useful in the development of immunoassays.3,4 This is one of the reasons for the increasing interest in adsorption studies of F(ab′)2 fragments onto different surfaces.5-8 The main purpose of our investigation is to achieve a better insight into the mechanism of adsorption of F(ab′)2 molecules onto different polymer surfaces. Some of the parameters that may influence the protein-surface interaction include electrostatic interactions, pH, surface charges, coadsorption of low molecular weight ions, hydrophobicity patches of protein and polymer, and the isoelectric point of the protein. In order to perform a widespread study of these factors, we have used six * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, May 15, 1996. (1) Nakamura, M.; Ohshima, H.; Kondo, T. J. Colloid Interface Sci. 1992, 149, 241. (2) Nakamura, R. M.; Tan, E. M. Hum. Pathol. 1978, 9, 85. (3) Bright, F. V.; Betts, T. A.; Litwiler, K. S. Anal. Chem. 1990, 62, 1065. (4) Ortega, J. L.; Ga´lvez, M. J.; Hidalgo-A Ä lvarez, R. Prog. Colloid Polym. Sci. 1995, 98, 233. (5) Kurosawa, S.; Kamo, N.; Muratsugu, M. Polym. Adv. Technol. 1991, 2, 253. (6) Kawaguchi, H.; Sakamoto, K.; Ohtsuka, Y.; Ohtake, T.; Sekiguchi, H.; Iri, H. Biomaterials 1989, 10, 225. (7) Buijs, J.; Lichtenbelt, J. W. Th.; Norde, W.; Lyklema, J. Colloids Surf. B 1995, 5, 11. (8) Ortega, J. L.; Hidalgo-A Ä lvarez, R. Biotechnol. Bioeng. 1995, 47, 633.
S0743-7463(95)01500-9 CCC: $12.00
polystyrene latexes with different superficial characteristics: nature of the surface ionic groups, surface charge density, sign of charge, and hydrophobic/hydrophilic character. Moreover, we have carried out adsorption and desorption experiments at different pHs to study the extent of the role played by electrostatic interactions. Afterward we performed an electrokinetic characterization of the F(ab′)2-latex complexes by means of electrophoretic mobility measurements. Finally, we have made a comparative study of the colloidal stability of these immunolatexes to check the viability of this system for developing immunoassays. 2. Experimental Section 2.1. Preparation of Latex Particles. All the latexes used were synthesized in our laboratory, employing styrene as majority monomer. The styrene (Merck) was previously distilled under low pressure (10 mmHg and 40 °C). There are two anionic latexes (JL1 and JL2) that have strong acid groups (sulfonate) in their surfaces, although they differ in surface charge density (σ0). Another couple of latexes (JL4 and JL7) are also anionic, the nature of their surface ionic groups being carboxyl. There are two more examples, a cationic latex (JL8) which has amidine groups and a hydrophilic polymer (JL10) synthesized with styrene and hydroxyethyl methacrylate (HEMA). Sulfonate latexes are styrene/sodium styrenesulfonate (Fluka) (St/NaSS) copolymers, which were prepared following the studies of different authors.9-11 The carboxyl latexes were synthesized using styrene as the only monomer. The carboxyl groups come from the initiator employed: 4,4′-azobis(4-cyanopentanoic acid) (ACPA) purchased from Aldrich. Synthesis conditions were fitted in accordance with the studies of Guthrie.12 The cationic latex was obtained using azo N,N′-dimethyleneisobutyramidine hydrochloride (AMDBA) as initiator, and only styrene was added as monomer in the polymerization reaction.13,14 Finally, the JL10 latex was a styrene/HEMA (Merck) copolymer synthesized in (9) Kim, J.-H.; Chainey, M.; El-Aasser, M. S.; Vanderhoff, J. W. J. Polym. Sci., Polym. Chem. Ed. 1989, 27, 3187. (10) de las Nieves, F. J.; Daniels, E. S.; El-Aasser, M. S. Colloids Surf. 1991, 60, 107. (11) Bastos, D.; de las Nieves, F. J. Colloid Polym. Sci. 1993, 271, 860. (12) Guthrie, W. H. New free radical initiators and their use in the preparation of polystyrene polymer colloids. Ph.D. Dissertation, University of Lehigh, 1985. (13) Hidalgo-A Ä lvarez, R.; de las Nieves, F. J.; van der Linde, A. J.; Bijsterbosch, B. H. Colloids Surf. 1986, 21, 259.
© 1996 American Chemical Society
3212 Langmuir, Vol. 12, No. 13, 1996
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Table 1. Synthesis Conditions of the Latexes: St, Styrene; NaSS: Sodium Styrenesulfonate; ACPA, 4,4′-Azobis(4-cyanopentanoic acid); ADMBA, Azo N,N′-dimethyleneisobutyramidine; HEMA, Hydroxyethyl Methacrylate latex
step
H 2O (mL)
St (g)
JL1 JL2
1 1 2 1 1 1 1 2
140 140 14 720 720 720 400 10
20.0 20.0 4.0 20.0 33.4 27.3 20.0 11.7
JL4 JL7 JL8 JL10
NaSS (mg)
K2S2O8 (mg)
NaHSO3 (mg)
NaHCO3 (mg)
86 86 344
182 182 15
71 71 12
94 94 19
ACPA (mg)
ADMBA (mg)
160 1480 102
HCl (ml)
HEMA (g)
stirring (r.p.m.)
temp (°C)
time (h)
3.0
35 35 35 350 350 350 350 350
45 45 45 80.6 80.6 50 70 70
9 7 10 20 22 24 1.5 7
96 372 488
260 100
NaOH (mg)
390
166
two steps. The polymerization reaction conditions of this coreshell latex were established by ourselves, after perusing the recipes used by some authors.15-17 All the latexes were prepared by emulsifier-free emulsion polymerization in a discontinuum reaction. The reactor was a spherical vessel (maximum volume ) 1 L), and solutions were stirred with a Teflon palette located at 1 cm over the bottom of the vessel. For the sulfonate latexes the reactor was different. They were synthesized in 0.5 L bottles which were bound to the top of an arm (15 cm) that can rotate vertically inside a thermostatic bath. Synthesis conditions are shown in Table 1. 2.2. Protein. F(ab′)2 antibody fragments from rabbit polyclonal IgG were kindly donated by Biokit S.A. (Spain). They were obtained by pepsin digestion of IgG, followed by gel filtration chromatography (Superose 12 HR 10/30, Pharmacia) and Protein-A chromatography, HiPAc (ChromatoChem), to remove undigested IgG. Purity was checked by SDS-Page electrophoresis. The isoelectric point (i.e.p.) of the F(ab′)2 molecules was determined by isoelectric focusing, and the i.e.p. values were found to be in the range 4.6-6.0. The molecular weight is 102 kD. 2.3. Adsorption Isotherms. F(ab′)2 concentrations before and after adsorption were determined by direct UV spectrophotometry at λ ) 280 nm (Σ ) 1.48 mL mg-1 cm-1), using a Milton Roy 601 Spectrophotometer. There were five experimental variables that remained constant in each adsorption experiment: total polymer area added (0.3 m2), final volume (10 mL), ionic strength (I ) 0.002), incubation temperature (25 °C), and incubation time (4 h). The adsorption experiments were carried out in a thermostatic bath where samples were gently agitated in a rotatory plate. Small amounts of the stock latex suspensions were added to different buffers containing F(ab′)2 from 0-400 µg/mL. Buffers used with the anionic latexes were acetate for pH’s 4 and 5, phosfate for pH’s 6 and 7, and borate for pH’s 8 and 9, with the cationic latex BIS-TRIS for pH’s 6 and 7, TRIS for pH’s 8 and 9, and AMP (2-amino-2-methyl-1-propanol) for pH 10. This was to avoid the specific adsorption of phosfate and borate ions which takes place when working with the cationic latex, as this could affect the electrostatic interaction between the F(ab′)2 fragments and the polymer surface. The same buffers mentioned above were also used both for the electrophoretic mobility measurements and for the colloidal stability studies. Once the incubation time had passed, samples were spun and supernatants were filtered using a polyvinylidene difluoride filter (Millipore, pore diameter ) 0.1 µm) before measuring the protein concentration. This last filter presents an extremely low affinity for protein adsorption, so the filtration step does not negatively interfere with the calculation of the adsorbed protein amounts. 2.4. Desorption Experiments. In order to study the reversibility or irreversibility of F(ab′)2 adsorption, we redispersed the sensitized latex particles (after the centrifugation step) in 10 mL of a certain buffered solution and then incubated them at 25 °C for 20 h. One can calculate the desorbed protein amount by repeating the centrifugation-filtration process. 2.5. Electrophoretic Mobility. Electrophoretic mobility measurements were performed with a Zeta-Sizer IV (Malvern (14) Galisteo, F.; Martı´n, A.; Hidalgo-A Ä lvarez, R. Colloid Polym. Sci. 1994, 272, 352. (15) Okubo, M.; Yamamoto, Y.; Uno, M.; Kamei, S.; Matsumoto, T. Colloid Polym. Sci. 1987, 265, 1061. (16) Tamai, H.; Hasegawa, H.; Suzawa, T. J. Appl. Polym. Sci. 1989, 38, 403. (17) Kondo, A.; Oku, S.; Higashitani, K. Biotechnol. Bioeng. 1991, 37, 537.
Instruments). The sensitized polymer particles were diluted in the desired medium. Then we took mobility data from the average of six measurements at the stationary level in a cylindrical cell. The standard deviation of such values was lower than 5%. 2.6. Colloidal Stability. The colloidal stability of the samples was spectrophotometrically studied using a wavelength of 570 nm. The critical coagulation concentration (ccc) at which latex particles rapidly coagulate was calculated measuring the variation of turbidity of a F(ab′)2-latex sample buffered at a determined pH. The turbidity of the sample increases when colloidal particles begin to aggregate upon mixing the latex suspension and a rich in KBr solution. The stability factor (W) is obtained as the ratio of two initial slopes of turbidity (τ) versus time experiments:
W)
(dτ/dt)f (dτ/dt)s
(1)
where “f” and “s” mean “fast” and “slow” coagulation rates, respectively. The ccc values are those points where the slope (d log W/d log C) reduces to zero.
3. Results and Discussion 3.1. Latex Particle Characterization. First, we carried out an exhaustive characterization of the latex particles in order to know the size of the microspheres and the solid content of the latex stocks, the surface charge density, and the hydrophobic/hydrophilic character. (a) Size of the Microspheres and Solid Content of the Latex Stocks. One must carefully determine these factors to perfectly know the amount of polymer area used in the adsorption experiments. This is why we determined the average particle diameter (L) using two different methods: (i) transmission electron microscopy (TEM) and (ii) photocorrelation spectroscopy (PCS) (System 4700c, Malvern Instruments). As can be seen in Table 2, the results obtained by both methods are very similar. TEM photographs showed the particles to be completely spherical and monodisperse. The average diameter of each latex sample was calculated sizing 691 particles for JL1 latex, 852 (JL2), 242 (JL4), 273 (JL7), 257 (JL8), and 344 (JL10). Therefore, the L values obtained by TEM are statistically significant. The polydispersity index (PDI) is quite near to 1, which means that all of them are highly monodisperse. It is assumed that a latex is monodisperse if the PDI < 1.05. (b) Surface Charge Density (σ0). This is one of the parameters that characterizes a colloidal system. The densities were determined by both conductometric and potentiometric titrations. The σ0 values obtained are also shown in Table 2. JL10 latex has two sort of superficial charged groups: (i) strong acid ones (sulfate) which come from the initiator used in its synthesis (persulfate) and (ii) weak acid groups (carboxyl) that come from the oxidation of some hydroxyl groups of HEMA molecules. The surface charge density may vary depending on the medium pH. This is why we have also studied this dependence by means of potentiometric titrations. Method
F(ab′)2-Coated Polymer Carriers
Langmuir, Vol. 12, No. 13, 1996 3213 Table 2. Main Characteristics of Latex Particles
latex
superficial polar groups
L PCSa (nm)
L TEMb (nm)
PDIc
σ0d (µC/cm2)
JL 1 JL 2 JL 4 JL 7 JL 8 JL 10
sulfonate sulfonate carboxyl carboxyl amidine hydroxyl, sulfate
187 ( 4 204 ( 5 342 ( 7 342 ( 5 198 ( 5 636 ( 16
185 ( 9 201 ( 11 331 ( 9 333 ( 7 183 ( 5 637 ( 14
1.014 1.013 1.002 1.002 1.002 1.002
-4.0 ( 0.3 -10.5 ( 0.7 -12.1 ( 0.2 -19.0 ( 0.4 +5.3 ( 0.2 -10.3 ( 0.4
ccce (pH 7) (mM KBr)
Triton X-100f (µmol/m2)
290 ( 20 700 ( 40 790 ( 40 1000 ( 100 160 ( 10
1.04 ( 0.19 1.06 ( 0.22 1.13 ( 0.23 1.52 ( 0.12 2.32 ( 0.07 0.56 ( 0.16
a L PCS: diameter obtained by photocorrelation spectroscopy. b L TEM: diameter obtained by transmission electron microscopy. c PDI: polydispersity index. d σ0: surface charge density. e ccc: critical coagulation concentration. f These data are directly related to the hydrophobic character of polymer surfaces.
a
b
angle of a sessile drop of water deposited on dried plugs made up of latex particles. However, we had to choose other analysis methods after finding several (and important) experimental problems. One of these methods was to adsorb a nonionic surfactant onto the polymer particles. Hydrophobic surfaces tend to adsorb high amounts of surfactant, while the hydrophilic ones only adsorb minor quantities.21,22 We adsorbed Triton X-100 onto our latexes, and the results are shown in Table 2. In the surfactant adsorption experiments, the results indicate that each surfactant molecule occupies 72 Å2 on cationic polystyrene latex (JL8), an average value of 140 Å2 on anionic polystyrene latexes (JL1-JL7), and 296 Å2 on the PSPHEMA copolymer latex (JL10). These different values of area per molecule are an indication of the different polymer polarities of the interface and suggest that the latexes used in this work have different hydrophobicities. Another technique we used to determine the hydration degree of the particles was viscosity measurements.16,23 The device employed was an automatic viscosimeter AVS 310 (Schott Gera¨te) using as measurement cell a capillary cell Ubbelohde 0c. The relationship between the specific viscosity of a latex solution and the volume fraction (φ) occupied by the polymer particles is given by
ηsp ) kf + k′′(kf)2φ φ Figure 1. (a) Surface charge density dependence on the pH for JL2 (9), JL4 (O), JL8 (2), and JL10 (]) latexes. (b) Electrokinetic charge density dependence on the pH for JL2 (9), JL4 (O), JL8 (2), and JL10 (]) latexes. The ionic strength of the medium was I ) 0.002.
details are described elsewhere.18 The σ0 values of four latexes versus pH are depicted in Figure 1a. The σ0 values for the JL1 (not shown) and JL2 latexes are independent of the pH, as they are samples that only have strong acid groups in their particle surfaces. Surface potential or, for that matter, the electrokinetic potential is more relevant than the surface charge density as a parameter to evaluate the electrostatic interaction between the F(ab′)2 molecules and the polymer surfaces. For that reason we have also calculated the ζ-potential of our latexes as a function of the pH. This has been made by means of electrophoretic mobility (µe) measurements. The µe data were converted into ζ-potential values by using the Dukhin-Semenikhin theory. The electrokinetic surface charge was calculated using the ζ-values. The σek values are represented in Figure 1b. Although σ0 and σek have different values, their trends with pH are quite similar, as can be seen in Figures 1. (c) Hydrophobic/Hydrophilic Character. It is wellknown that one of the most important driving forces that acts during the protein adsorption process is the hydrophobic one.19,20 Initially we tried to measure the contact (18) Bastos, D.; Ortega, J. L.; de las Nieves, F. J.; Hidalgo-AÄ lvarez, R. J. Colloid Interface Sci. 1995, 176, 232.
(2)
where k is a numerical factor calculated by Einstein which is equal to 2.5 for spherical particles and f is a factor related to the hydration layer (∆) of the particles. In spheres ∆ is equal to
[(
) ]
Fs ∆ ) r[f1/3 - 1] ) r δ + 1 Fo
1/3
-1
(3)
where r is the particle ratio, Fs is the polystyrene density (1.054 g/cm3), Fo is the solvent density (0.997 g/cm3 for water at 20 °C), and δ is the grams of water that soak a gram of solute. The viscosity results are shown in Figure 2, and the ∆ values obtained using eq 3 are (in nm) 5.5 ( 0.8 (JL1), 6.1 ( 0.5 (JL2), 7.5 ( 3.0 (JL4), 4.1 ( 2.5 (JL7), and 14.5 ( 0.8 (JL10). The hydrophobicity of latex JL8 has been established by only one method, as it was impossible to measure the viscosity. After analyzing all the results, it can be claimed that the most hydrophobic latex is the cationic latex (JL8), the most hydrophilic latex is JL10, and the other samples (JL1, JL2, JL4, and JL7) have, more or less, the same hydrophobic/hydrophilic character. The presence of poly(19) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267. (20) Norde, W.; Lyklema, J. J .Biomater. Sci., Polym. Ed. 1991, 2, 183. (21) Patxon, T. R. J. Colloid Interface Sci. 1969, 31, 19. (22) Brouwer, W. M.; Zsom, R. L. J. Colloids Surf. 1987, 24, 195. (23) Suzawa, T.; Shirihama, H. Adv. Colloid Interface Sci. 1991, 35, 139.
3214 Langmuir, Vol. 12, No. 13, 1996
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HEMA chains on the JL10 surface explains why its surface is rather hydrophilic. Why does JL8 present the most hydrophobic polymer surface of all the (homo)polystyrene latexes? This seems to be because the polar groups of this latex (amidine) are placed in an organic heterocycle, and as such they are in a more apolar environment than the sulfonate or carboxyl groups of the rest of the latexes. Holgado et al.24 have confirmed this result measuring contact angles on latex samples synthesized with the same initiator as used by us. 3.2. F(ab′)2 Adsorption Isotherms. First, we carried out an adsorption isotherm of F(ab′)2 onto each latex sample at pH 7. Adsorbed amounts (Γads) versus the protein concentration in solution after adsorption (Ce) are shown in Figure 3. We can highlight two different aspects of these experiments: (i) Once the surface is totally covered by a F(ab′)2 layer, the adsorbed amount remains constant in a well defined “plateau” value (Γpl). (ii) The slope of the initial part of each isotherm curve qualitatively reflects the affinity of the protein for the surface. It can be seen that a high affinity exists in the adsorption of F(ab′)2 onto the JL8 (highly hydrophobic) latex. Next we want to comment on some interesting aspects. With regard to the dimensions of the F(ab′)2 fragments (142 × 38 × 38 Å3) obtained from structures of crystallized IgG molecules,25 a monolayer of F(ab′)2 molecules would be equal to 3.2 mg/m2 when adsorbed in a “side-on” orientation. Nevertheless, we have obtained Γpl values up to 6.6 mg/m2 (in JL8 latex). This result does not mean that F(ab′)2 molecules form a bilayer on the cationic surface, as these protein fragments can be adsorbed in
different orientations. Recently Buijs et al.7 have postulated theoretical values for the F(ab′)2 monolayer. They range from 2.0 mg/m2 (“side-on” orientation for molecules with totally opened arms) to 8.7 mg/m2 for an “end-on” orientation. It should be noticed that F(ab′)2 fragments still have the hinges that are in the original IgG molecules which they come from. For the sulfonate latexes, the adsorbed amounts and the affinity for protein adsorption are lower for the most charged latex (JL2) than for the lesser charged one (JL1). This can be explained by the electrostatic repulsion that exists between the anionic polymer surface and the negatively charged protein fragment molecules. A similar result is found for the carboxyl latexes, where the adsorbed F(ab′)2 amounts also are lower for the most charged latex (JL7). The most striking results are obtained for both the highly hydrophilic (JL10) and hydrophobic (JL8) latexes. The small amounts of F(ab′)2 adsorbed on the JL10 latex cannot be explained taking only into account the electrostatic repulsion, since σ0 values for both JL2 or JL7 are higher. On the other hand, both the high affinity of the protein for the cationic surface and the high amount of F(ab′)2 molecules adsorbed on the JL8 latex do not seem to be explained by electrostatic attraction forces, since JL8 is a relatively low charged latex. From this one might conclude that hydrophobic forces must play a very important role in protein adsorption processes. This suggests an adsorption mechanism of F(ab′)2 molecules onto the hydrophobic patches of polystyrene, caused not only by the dehydration of hydrophobic side groups of the polypeptide chains but also mainly by the dehydration of the hydrophobic polystyrene surface. This process is almost completely due to the entropy increase caused by the release to the bulk of the structured water in contact with hydrophobic components.19 This is why F(ab′)2 adsorbs onto hydrophobic latexes even under electrostatic repulsion conditions. As we have used only one protein but different polymer surfaces, it is obvious that the differences found in the adsorption isotherms are exclusively due to the nature of these surfaces. Moreover, the adsorbent composition determines the extent of the structural rearrangements that take place when protein molecules adsorb at the interface.26 Some authors27,28 have also demonstrated the great influence of the hydrophobic force on protein adsorption phenomena. Subsequently, we studied the maximum adsorbed amount of F(ab′)2 as a function of the medium pH. Results are presented in Figures 4 (solid lines). From these experiments we can highlight the following conclusions: (i) In the anionic latexes the maximum value for Γpl is achieved around pH 5 (slightly lower pH than the average i.e.p. of the F(ab′)2 ) 5.3). At this pH the macromolecules present a compact conformation, since the biopolymer is almost uncharged (really, many of its polar groups are charged but the net charge of the whole molecule is zero or near zero). Therefore, as at such a pH the protein usually attains a high structural stability, the area occupied by an adsorbed molecule will be less than that it would cover when the adsorption pH is different from the protein i.e.p. Moreover, the intermolecular repulsions between the adsorbed molecules are also low at this pH. These two factors contribute to reach a maximum value of Γpl near the i.e.p. of the F(ab′)2. In fact, there are numerous authors who have obtained similar behaviors when working with different proteins.29-32
(24) Holgado, J. A.; Martı´n, A.; Martı´nez, F.; Cabrerizo, M. A. Proceedings of the Solids/Fluid Interfaces Capillarity and Wetting Congress, September 1-6, Arnhem; 1992. (25) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 216, 3753.
(26) Bale, M. D.; Danielson, S. J.; Daiss, J. L.; Goppert, K. E.; Sutton, R. C. J. Colloid Interface Sci. 1989, 132, 176. (27) Tilton, R. D.; Robertson, Ch. R. Langmuir 1991, 7, 2710. (28) Warkentin, P.; Wa¨livaara, B.; Lundstro¨m, I.; Tengvall, P. Biomaterials 1994, 15, 786.
Figure 2. Linear behavior of ηsp/φ versus φ for JL1 (3), JL2 (9), JL7 (O), and JL10 ([) latexes.
Figure 3. F(ab′)2 adsorption isotherms at pH 7 (I ) 0.002) for JL1 (0), JL2 (9), JL4 (O), JL7 (b), JL8 (2), and JL10 (]) latexes.
F(ab′)2-Coated Polymer Carriers
Figure 4. (a) Maximum amount of adsorbed F(ab′)2 as a function of adsorption pH for JL1 (O) and JL2 (9) latexes (solid lines). Amounts that remain adsorbed after resuspending the above samples in pH 8 (segmented lines). Vertical dotted line represents the average F(ab′)2 i.e.p. value. (b) Maximum amount of adsorbed F(ab′)2 as a function of adsorption pH for JL4 (b) and JL7 (0) latexes (solid lines). Amounts that remain adsorbed after resuspending the above samples in pH 8 (segmented lines). Vertical dotted line represents the average F(ab′)2 i.e.p. value. (c) Maximum amount of adsorbed F(ab′)2 as a function of adsorption pH for JL8 (2) and JL10 (]) latexes (solid lines). Amounts that remain adsorbed after resuspending the JL10 samples in pH 8 (segmented lines). Vertical dotted line represents the average F(ab′)2 i.e.p. value.
(ii) For JL8 latex the pH of maximum adsorption is shifted to pH 7. This confirms that in media of low ionic strength the electrostatic interaction also plays an important role in the protein adsorption process. It should be noted that for anionic latexes (especially for JL2 and JL10) there is a higher adsorption at pH 4 (where macromolecules posses a positive net charge) than at pH 6 (upper (29) MacRitchie, F. J. Colloid Interface Sci. 1972, 38, 484. (30) Morrisey, B. N.; Stromberg, R. R. J. Colloid Interface Sci. 1974, 46, 152. (31) Bagchi, P.; Birnbaum, S. M. J. Colloid Interface Sci. 1981, 83, 460. (32) Koutsoukos, P. G.; Numme-Young, C. A.; Norde, W.; Lyklema, J. Colloids Surf. 1982, 5, 93.
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limit of the F(ab′)2 i.e.p. range). Therefore, the maximum adsorbed amount is obtained near the i.e.p. of the protein but slightly shifted toward a more acid pH for a negatively charged polymer surface and toward a more basic pH for a positively charged one. Similar results have recently been obtained by other authors.7 (iii) If the electrostatic attractive forces are responsible for the high Γpl values achieved at acid pH’s (3 and 4) in the case of the most charged sulfonated latex (JL2), why is there a different behavior for the carboxyl latexes? The answer can be found in Figure 1. As pH decreases, the charge of this kind of latexes is drastically reduced, and so the attractive interaction at pH’s 3 and 4 is quite small. To corroborate the above conclusions, we redispersed all the anionic latex samples in pH 8 (I ) 0.002), where F(ab′)2 and polymer surfaces have the same sign of charge. The amount of F(ab′)2 that still remains adhered on the colloidal particles is also shown in Figure 4 (segmented lines). The differences among the original samples (solid lines) and those redispersed in pH 8 give an idea about the electrostatic attractive contribution during the F(ab′)2 adsorption process. As can be seen, most of the adsorption of protein molecules onto the hydrophilic latex (JL10) at pH’s 5, 4, and 3 is due to electrostatic interactions. The amount of desorbed protein (in those samples for which incubation was carried out at pH’s around and lower than the protein i.e.p.) is higher for the most charged latexes. JL2 (sulfonated) and JL7 (carboxyl) samples release more F(ab′)2 molecules than JL1 (sulfonated) and JL4 (carboxyl) latexes, respectively. (iv) As adsorption pH is far from the protein i.e.p., the molecules are more distended due to internal electrostatic repulsions. Therefore, when the protein is near to its sorbent, it will occupy a more extensive area than in the i.e.p. case. It might be expected that the configurational change between the macromolecules in the bulk and those spreaded on the polymer surface would not be very large. However, even under these conditions F(ab′)2 adsorption takes place. One can justify this result on the basis of the dehydration of the hydrophobic patches of polystyrene particles. This produces a total entropy increase caused by the liberation of the initial structured water molecules. This is why at pH 9 the hydrophilic latex (JL10) only adsorbs minor F(ab′)2 amounts. Different authors7,23 obtain similar results when working with hydrophilic latexes. Besides, there is another effect that contributes to the decrease of the Γpl values at both sides of the F(ab′)2 i.e.p. This is the lateral interaction (mainly repulsive) between the molecules adhered at the water/polymer interface. This interaction is energetically considerable, as Jonhson et al. have recently shown.33 3.3. Desorption Experiments. Although in many cases the protein adsorption can be considered as an irreversible process, there are some systems where reversibility can occur. It must be noted that such a reversibility largely depends on the protein structure.19 For “hard proteins” (such as lysozyme, chymotrypsinogen, ribonuclease, R-lactoalbumine, etc) the adsorption process could be reversible. However, the F(ab′)2 fragment belongs to the “soft protein” group (IgG, hemoglobin, BSA, etc.) for which the adsorption process seems to be irreversible. In order to check this item, we carried out two sorts of experiments. On the one hand, we redispersed the samples used for the adsorption isotherms (Figure 3) in the same medium as was used during initial incubation. We found that no desorption took place in these dilution experiments, at least during the observation period. Therefore, the F(ab′)2 adsorption on polystyrene surfaces (33) Johnson, C. A.; Wu, P.; Lenhoff, A. M. Langmuir 1994, 10, 3705.
3216 Langmuir, Vol. 12, No. 13, 1996
can be considered as an irreversible process. On the other hand, we redispersed some samples under different conditions, as we describe below: Once the colloidal particles were sensitized at two pH’s (5 and 7), each sample was redispersed for 20 h at (a) pH 5 and I ) 0.002, (b) pH 8 and I ) 0.002, and (c) the same initial pH but at high ionic strength (I ) 0.25). The latter experimental conditions eliminate the electrostatic interactions among protein molecules and polymer surfaces, due to the charge screening provoked by a high salt concentration. Results are depicted in Figure 5. Although these experiments were performed for every latex, we thought it more than enough to show only four of them (a sulfonate, a carboxyl, the cationic, and the hydrophilic latex). The most striking results are as follows: (i) When redispersing in a high ionic strength medium, one always obtains a non-negligible F(ab′)2 desorption. This feature confirms the importance of the electrostatic attractive interaction during the adsorption step in low ionic strength media. If one completely removes this contribution, the protein amount that still remains adhered on the hydrophilic latex (Figure 5d) is almost zero. This fact does not indicate that F(ab′)2 is a “hard protein”. What happens is that when a protein is adsorbed on a hydrophilic surface, it does not tend to spread itself, exposing its inner hydrophobic patches facing the sorbent surface. Therefore, although F(ab′)2 is a “soft protein”, as we previously mentioned, it might be thought that in this case such a fragment behaves as a hard protein, since there is almost no hydrophobic interaction between JL10 polymer and F(ab′)2 molecules. On the other hand, it can be seen that the high amount of F(ab′)2 initially adsorbed onto the cationic (and most hydrophobic) latex depends strongly on the electrostatic forces. At least, surface and macromolecules have a different sign of charge at pH 7, and a great part of the initial adsorbed protein (33% approximately) is released when the ionic strength of the medium is increased. Nevertheless, after the desorption experiments were finished the protein coverage in JL8 particles was extremely high due to the hydrophobicity of such a polymer surface. (ii) With a desorption pH nearer to the F(ab′)2 i.e.p. than the initial adsorption pH, there is practically no protein desorption. However, with a desorption pH further from the F(ab′)2 i.e.p. than the initial adsorption pH there is a slight desorption caused by repulsion interactions between protein-protein molecules and protein-surface. (iii) A negligible desorption takes place in samples with low protein coverage. 3.4. Electrophoretic Mobility. The electrokinetic behavior of colloidal particles covered by F(ab′)2 depends on (a) the amount of adsorbed protein and both (b) the pH and (c) the ionic strength of the redispersion medium. We can start discussing the electrophoretic mobility (µe) measurements versus the F(ab′)2 coverage, once the pH and ionic strength (pH 7, I ) 0.002) are fitted. Results are shown in Figure 6. The most striking result is that in anionic latexes the µe decreases (in absolute value) as protein coverage increases. At pH 7, F(ab′)2 molecules have a negative net charge. Despite that, the mobility becomes less negative. This can be justified by two nonexclusive phenomena. (i) It is assumed that coadsorption of low molecular weight ions takes place in protein adsorption processes.19,34 Therefore, these ions (located between the polymer surface and the adsorbed macromolecule layer) will affect the electrophoretic mobility of the system.35 Elgersma et (34) Norde, W. Cells Mater. 1995, 5, 97.
Ortega Vinuesa et al.
Figure 5. F(ab′)2 amounts that remain adsorbed on latex particles after performing three different desorption experiments. Left: initial adsorption was carried out at pH 5. Right: initial adsorption was carried out at pH 7. (a) JL1; (b) JL7; (c) JL8; (d) JL10.
al.36,37 and Martı´n et al.38 use the following equation in order to quantify the amount of adsorbed ions at the polystyrene-BSA interface: (35) Shirahama, H.; Takeda, K.; Suzawa, T. J. Colloid Interface Sci. 1986, 109, 552. (36) Elgersma, A. V.; Zsom, R. L. J.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1990, 138, 145. (37) Elgersma, A. V.; Zsom, R. L. J.; Norde, W.; Lyklema, J. Colloids Surf. 1991, 54, 89. (38) Martı´n, A.; Cabrerizo, M. A.; Hidalgo, R. Colloids Surf. 1994, 92, 113.
F(ab′)2-Coated Polymer Carriers
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Figure 6. Electrophoretic mobility of JL1 (0), JL2 (9), JL4 (O), JL7 (b), JL8 (2), and JL10 (]) latex particles as a function of F(ab′)2 coverage. Experimental conditions: pH 7, I ) 0.002.
∆adsσek ) σek(PS-protein) - σek(PS) - σek(protein)ΓadsA
(4)
where σek is the charge density per unit area at the slipping layer of the covered polystyrene particles (PS-protein), bare latex(PS), and dissolved protein (protein); A is the surface area of protein per unit mass.
σek )
4n0ze zeζ sinh κ 2kT
( )
(5)
where n0 is the bulk concentration of ions, z is the ion valency, e is the elementary charge, κ-1 is the doublelayer thickness, k is the Boltzmann constant, T is the absolute temperature, and ζ is the electrokinetic potential. However, we could not apply eq 4, since the σek(F(ab′)2) value is still unknown. Elgersma36 was also unable to calculate ∆adsσek for the polystyrene-monoclonal IgG interface for the same reason. (ii) One must keep in mind that µe reflects the ζ-potential of the colloidal particles but not the superficial potential Ψ0 (directly related to σ0). As a smooth polymer surface is being covered by macromolecules, it becomes more ridged and irregular. This shifts the slipping plane outward causing a decrease in ζ-potential and thus decreasing the µe value. Subsequently, we carried out the mobility experiments versus the redispersion pH (I ) 0.002). The most representative results are shown in Figure 7. In these plots the µe values of bare latex particles, half-covered particles, and totally-covered particles are depicted. In all cases the i.e.p. of the F(ab′)2-latex complexes tends to the i.e.p. of the pure protein (5.3) as colloidal particles have higher amounts of adhered F(ab′)2. Nevertheless, as can be seen, the electrokinetic behavior of these complexes clearly depends on the nature of the polymer adsorbent. The maximum value of Γpl (Figure 4) is obtained around the i.e.p. of the F(ab′)2-latex complexes (Figures 7), instead of around the i.e.p. of the pure protein. At these pH’s (which are not far from the F(ab′)2 i.e.p.) protein molecules have a compact structure and also there is an additional electrostatic attraction force that favors the protein-surface union. We have performed a deeper analysis of the above results. Figure 7a shows the mobility data obtained with the JL1 (sulfonated) and JL8 (cationic) latexes. As both polymers have approximately the same σ0 value (although a different sign of charge), we can suppose that the electrophoretic mobility of the F(ab′)2 molecules (in the absence of latex particles) corresponds to the segmented line of the figure. This hypothetical mobility has been obtained averaging the µe values of the JL1 particles totally
Figure 7. (a) Electrophoretic mobility versus pH of JL1 latex (0), JL1-F(ab′)2 (1.7 mg/m2) (b), JL1-F(ab′)2 (3.0 mg/m2) (3), JL8-F(ab′)2 (3.8 mg/m2) ([), JL8-F(ab′)2 (1.6 mg/m2) (]), and JL8 latex (2). Dotted line: hypothetical electrophoretic mobility of F(ab′)2 molecules. (b) Electrophoretic mobility versus pH of JL2 latex (9), JL2-F(ab′)2 (1.1 mg/m2) (O), and JL2-F(ab′)2 (2.9 mg/m2) ([). (c) Electrophoretic mobility versus pH of JL7 latex (9), JL7-F(ab′)2 (1.1 mg/m2) (O), and JL7-F(ab′)2 (2.8 mg/m2) ([).
covered by F(ab′)2 fragments and the JL8 particles totally covered by the same protein. Although this assumption could be questionable, one can check in the last figure that the i.e.p. of the protein calculated in this way exactly coincides with the average i.e.p. of the F(ab′)2, which is equal to 5.3. Therefore, this method allows us to calculate σek(F(ab′)2). We converted µe into ζ-potential values using the Dukhin-Semenikhin theory.39 Later, we calculated the σek values for the latexes totally covered by F(ab′)2 (σek(PS-F(ab′)2)), for the bare latex (σek(PS)), and for the protein (σek(F(ab′)2)), and then we determined the ∆adsσek values, which are shown in Figure 8. The A value (eq 4) was calculated on the basis of the dimensions of the F(ab′)2 fragments.25,40 This last figure gives information about the low molecular weight (MW) ion participation in the (39) Dukhin, S. S.; Semenikhin, N. M. Kolloidn. Zh. 1970, 31, 36. (40) Marquart, M.; Deisenhofer, J.; Huber, R.; Palm, W. J. Mol. Biol. 1980, 141, 369.
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a
Figure 8. Superficial density of low MW ions adsorbed onto JL1 (0), JL2 (9), JL4 (O), JL7 (b), and JL8 (2) particles totally covered by F(ab′)2 as a function of medium pH.
F(ab′)2 adsorption process. As we have already pointed out, because of overall electroneutrality ∆adsσek represents the charge transfer between solution and the adsorbed layer. It can be checked that the maximum amount of cations adsorbed in the interface of PS--protein occurs at basic pH’s (8 and 9). At these pH’s, both particles and F(ab′)2 molecules are negatively charged, and thus a coadsorption of positive ions is needed to considerably diminish the electrostatic repulsion among the polymer surface and the protein. As the medium is changed to a more acid one, F(ab′)2 becomes less charged and the adsorption of cations decreases. When pH coincides with the protein i.e.p. (5.3), there is still a slight adsorption of such ions. This coadsorption process reduces to zero when particles and F(ab′)2 fragments have a different sign of charge, i.e. pH ≈ 4.5 for sulfonated latexes and pH ≈ 4.9 for carboxyl latexes. If protein molecules become more positively charged, then there will be a slight coadsorption of anions. As can be seen, the results found in anionic latexes are very similar, but a different behavior is obtained with JL8 samples. Nevertheless, we could explain these ∆adsσek values using the same reasoning. At low pH (i.e. 4) there is a high adsorption of anions to compensate the proteinpolymer electrostatic repulsion, as both of them are positively charged. In this case, the pH where there is no low MW ion coadsorption is higher than the F(ab′)2 i.e.p. For upper pH values, an adsorption of cations takes place. However, we have not been able to find out a reasonable argument to explain the high ∆adsσek values obtained at pH’s 8 and 9 in the JL8-F(ab′)2 complexes. 3.5. Correlation between Colloidal Stability and Mobility of Sensitized Particles. Before studying the stability of our samples we performed the electrophoretic mobility measurements against the ionic strength of the redispersion medium, pH being neutral. The main reason why these experiments were carried out is because both stability results and electrokinetic behavior must be correlated in colloidal systems, so the DLVO theory predicts. In this case, our protein-latex complexes also had two different F(ab′)2 loads: half and total coverage. The most important results are shown in Figure 9. As can be ssen, the µe values decrease (in absolute value) as latex colloidal microspheres are covered by protein. Moreover, the µe maximum is attenuated (and even disappears) when latex particles are coated by F(ab′)2 molecules. The explanation of such a behavior can be found taking into account the studies of Ohshima et al.41 However, our interest is focused on relating these results with those obtained by stability experiments. (41) Ohshima, H.; Nakamura, M.; Kondo, T. Colloid Polym. Sci. 1992, 270, 270.
b
c
d
Figure 9. (a) Electrophoretic mobility of JL1 latex (9), JL1F(ab′)2 (1.7 mg/m2) (O), and JL1-F(ab′)2 (3.0 mg/m2) ([) samples versus ionic strength. (b) Electrophoretic mobility of JL2 latex (9), JL2-F(ab′)2 (1.1 mg/mm) (O), and JL2-F(ab′)2 (2.9 mg/m2) ([) samples versus ionic strength. (c) Electrophoretic mobility of JL4 latex (9), JL4-F(ab′)2 (1.4 mg/m2) (O), and JL4-F(ab′)2 (3.1 mg/m2) ([) samples versus ionic strength. (d) Electrophoretic mobility of JL8 latex (9), JL8-F(ab′)2 (1.6 mg/m2) (O), and JL8-F(ab′)2 (3.8 mg/m2) ([) samples versus ionic strength.
As we mentioned in the Introduction, one of the possible advantages of using F(ab′)2 fragments instead of the whole IgG molecule is that colloidal particles covered by polyclonal IgG (i.e.p. range ) 6.5-8.0) are normally unstable under physiological conditions (pH ) 7.4, I ) 0.15). This
F(ab′)2-Coated Polymer Carriers
a
Langmuir, Vol. 12, No. 13, 1996 3219
a
b b
c Figure 10. (a) Dependence of the stability factor (W) on the electrolyte concentration for JL2 latex (9), JL2-F(ab′)2 (1.1 mg/m2) (O), and JL2-F(ab′)2 (2.9 mg/m2) ([) samples. (b) Dependence of the stability factor (W) on the electrolyte concentration for JL8 latex (9), JL8-F(ab′)2 (1.6 mg/m2) (O), and JL8-F(ab′)2 (3.8 mg/m2) ([) samples.
is a serious problem while developing diagnosis tests based on particle enhanced immunoassays. Nevertheless, as the F(ab′)2 i.e.p. is more acidic (i.e.p. range ) 4.7-6.0), the instability problem could be solved. This is why we have proceeded to determine the ccc of our samples. In Figure 10, the dependence of the stability factor (W) on the electrolyte concentration ([KBr]) is shown for JL2 and JL8 latexes. The ccc values depend on the F(ab′)2 coverage. Besides, colloidal stability varies as a function of the redispersion pH. Figure 11 shows the ccc values for our complexes when they have different protein loads. These experiments were carried out at different pH’s. Some of the ccc values are shown in Table 3. The results can be explained in the following way: (i) The slight amount of F(ab′)2 adsorbed on the hydrophilic latex (JL10) does not change the ccc with regard to the bare polymer particles. (ii) For the rest of the anionic latexes, the ccc of the protein-polystyrene complexes decreases very sharply, as colloidal particles begin to be covered by F(ab′)2. If Γads is higher than a certain limit value (≈1.5 mg/m2), the ccc of these complexes keeps a constant value. These last samples are more unstable at pH 5. The electrophoretic mobility of these protein-latex particles (Figures 7) is near zero at this acid pH. F(ab′)2-JL2 complexes had a high value of µe (in absolute value) at pH 5, and as can be seen, they also possess the highest ccc values (Table 3). The stability results of F(ab′)2-JL1 and F(ab′)2-JL4 samples at pH 4 also can be explained taking into account the Figure 7 data. Nevertheless, when the redispersion medium has a neutral pH, the above plots would not explain the low values of ccc found. To understand it, one must pay
d
Figure 11. (a) Ccc of JL1-F(ab′)2 complexes versus protein coverage: pH 4 (0); pH 5 ([); pH 7 (O); pH 9 (2). (b) Ccc of JL2-F(ab′)2 complexes versus protein coverage: pH 5 ([); pH 7 (O); pH 9 (2). (c) Ccc of JL4-F(ab′)2 complexes versus protein coverage: pH 4 (0); pH 5 ([); pH 7 (O); pH 9 (2). (d) Ccc of JL8-F(ab′)2 complexes versus protein coverage: pH 5 ([); pH 7 (O); pH 9 (2).
attention to Figure 9, where the electrophoretic mobility of the totally covered particles decreases as ionic strength increases. At I ) 0.10 there are great differences between the mobility values of bare latex and the F(ab′)2-latex complexes. This feature could be understood if there is a flux of counterions toward the solid-water interface.
3220 Langmuir, Vol. 12, No. 13, 1996
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Table 3. Ccc Values of Some F(ab′)2-Latex Complexes with High Protein Coverage sample
medium pH
ccc (mM in KBr)
JL1-F(ab′)2 (2.9 ( 0.1 mg/m2) JL2-F(ab′)2 (2.5 ( 0.5 mg/m2) JL4-F(ab′)2 (3.0 ( 0.2 mg/m2) JL7-F(ab′)2 (2.6 ( 0.2 mg/m2) JL8-F(ab′)2 (4.5 ( 0.2 mg/m2) JL10-F(ab′)2 (1.0 ( 0.1 mg/m2)
pH 5 pH’s 7 and 9 pH 5 pH’s 7 and 9 pH 5 pH’s 7 and 9 pH 5 pH’s 7 and 9 pH 5 pH’s 7 and 9 pH 5 pH’s 7 and 9
27 ( 4 55 ( 5 100 ( 10 100 ( 10 20 ( 3 60 ( 5 70 ( 3 95 ( 5 35 ( 2 7(4 63 ( 5 135 ( 10
These ions could diffuse across the adsorbed protein layer, leading to a ζ-potential diminution. This is why the stability of the F(ab′)2-latex particles would be so low. (iii) For the cationic latex we found similar results. As particles are being covered by F(ab′)2, their stability decrease. However, they are more stable at pH 5 than at pH 7 or 9. Although at pH 5 the external protein layer is practically uncharged, the global behavior is affected by the nature of the polymer surface, as we previously demonstrated. The mobility of these samples is positive at pH 5, negative at pH 9, and zero around neutral pH. The trend of variation of these data agrees with these results obtained from stability experiments. (iv) A rapid aggregation of the JL4 (at pH 4) and JL8 (at pH 9) latexes occurs when they are partially covered by F(ab′)2. Protein molecules and polymer surfaces are oppositely charged, and the system tends to agglutinate by a bridging process.42 (v) Although the F(ab′)2-latex samples would not be stable under physiological conditions, the stability of these immunocomplexes is higher than that of those obtained when adsorbing IgG. This we have previously demonstrated43,44 adsorbing polyclonal F(ab′)2 and IgG onto the same latex sample (a sulfate one). Nevertheless, the (42) Singer, J. M.; Vekemans, F. C. A.; Lichtenbelt, J. W. Th.; Hesselink, F. Th.; Wiersema, P. H. J. Colloid Interface Sci. 1973, 45, 608. (43) Serra, J.; Puig, J.; Martı´n, A.; Galisteo, F.; Ga´lvez, M. J.; HidalgoA Ä lvarez, R. Colloid Polym. Sci. 1992, 270, 574. (44) Ortega, J. L.; Hidalgo-A Ä lvarez, R. Colloids Surf. 1993, 1, 365.
stability of our F(ab′)2-latex systems can be increased if this protein and BSA are sequentially adsorbed onto the polystyrene particles.45 4. Conclusions The main conclusions are summarized above. (1) Before adsorbing proteins onto different polymerwater interfaces it is very important to carry out an exhaustive characterization of those interfaces. This will allow a correct interpretation of the obtained results. This is why it would be advisable to determine particle sizes, σ0 values, and hydrophobicity degree using, at least, two different techniques. (2) The main driving force in the F(ab′)2 adsorption onto polymer surfaces is the hydrophobic one. Nevertheless, if adsorption isotherms are performed in media of low ionic strength, electrostatic interactions play an important role. This is why the maximum amounts of adsorbed protein are obtained around the i.e.p. of the F(ab′)2-latex complexes. (3) The protein-surface affinity depends on the hydrophobicity of the adsorbent. The F(ab′)2 adsorption onto hydrophobic surfaces can be considered as an irreversible process. (4) The stability of colloidal particles covered by polyclonal F(ab′)2 is not very high. Nevertheless, the use of the F(ab′)2 fragment improves this colloidal stability in comparison with those latex particles sensitized by IgG. Moreover, the use of the F(ab′)2 moieties would avoid erroneous diagnosis if particle-enhanced immunoassays are carried out in media where there was a slight presence of rheumatoid factors. Acknowledgment. This work has been financial supported by the “Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT)” MAT-0560/94. We would also like to thank Biokit S.A. (Barcelona, Spain) for purifying, characterizing, and supplying the F(ab′)2 fragments. Finally, we would like to express our gratitude to Herman van Bellingen for correcting the final English version. LA951500M (45) Ortega, J. L.; Molina, J. A.; Hidalgo-A Ä lvarez, R. J. Immunol. Methods 1996, 190, 29.
