Electrokinetics

To determine the role of electrostatic interactions in IgG adsorption, several authors [36-38,40,42,43] have studied the electrokinetic behavior of IgG-coated surfaces. Furthermore, the electrophoretic mobility of IgG-coated surfaces can be suitable to predict the colloidal stability of these systems. Figure 8 shows the electrophoretic mobility of IgG-coated polystyrene beads (IgG-PS) as a function of the amount of adsorbed IgG (r). With increasing r the absolute value of mobility decreases to reach a plateau value. This decrease is dependent on the pH, i.e., on the charge of the IgG molecules. In Fig. 9 mobilities at complete coverage of the IgG-PS complexes are given as a function of resuspension pH

FIG. 8 Electrophoretic mobility vs. adsorbed amount of rabbit IgG at 2 mM NaCl and three different adsorption-resuspension pH values: pH 5 (•), pH 7 (▲), pH 9 (■).

for both types of charged surfaces. There is a significant difference between IgG adsorbed on negatively and positively charged surfaces. This difference can be related to electrostatic interactions between IgG and the charged surface. The IEPs of the polyclonal IgG-PS complexes are 6 and 8 for the anionic and cat-ionic PS beads, respectively. This difference indicates that the surface charge must compensate, at least partly, the charge of the IgG molecules. This effect has also been seen with monoclonal antibodies on positively and negatively charged surfaces [36,38]. Also, it should be noted that, in both cases, the mobility of the IgG-PS complexes is decreased in comparison with the mobility of the bare PS beads, which could explain the extremely low colloidal stability of the polyclonal IgG-coated surfaces. The structure of the electrical double layer (EDL) of polyclonal IgG-coated polystyrene beads has been studied by Galisteo et al. [49]. The main conclusions drawn from these studies are (1) that ions in the electrical double layer surrounding the IgG-polymer surface (especially those under the hydrodynamic slipping plane) have a greater ionic mobility when the electric charge in the protein molecule has the same sign as the electric

FIG. 9 Electrophoretic mobility vs. resuspension pH: bare cationic PS beads (•), saturated cationic PS beads (O), bare anionic PS beads (■), saturated anionic PS beads (□).

groups in the particle surface; and (2) that the anomalous surface conduction mechanism is more pronounced in this case in the surface charge region.

A different approach to the electrophoretic mobility of antibody-carrying latex particles has been used by Nakamura et al. [50,51]. According to these authors, the Z potential loses its meaning for the IgG-latex particles, since the electrophoretic mobility is insensitive to the precise position of the slipping plane. Upon analysis, they conclude that the depth of the bound IgG layer varied from 3.5 to 8.5 nm, and it decreased with increasing ionic strength, suggesting that conformation of the bound IgG becomes more compact following addition of electrolyte. It is considered that the addition of excessive electrolyte ions reduces the intramolecular and intermolecular electrostatic interaction of the IgG molecules bound to the surface of latex particles. This new approach to the interpretation of the electrophoretic mobility even provides conformational information about the IgG that is fixed on polymer surfaces, which is its major advantage against the classical electrical double-layer theory.

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