Immunoglobulin Adsorption

The literature concerning the study of immunoglobulin G adsorption at solidliquid interfaces has a long and confusing history [20,27,28]. We note specifically that (1) experimental adsorption isotherms performed in different laboratories on quite similar systems often conflict; and (2) minor changes in experimental conditions (pH, ionic strength, temperature) may result in major differences in the measured adsorption. These studies are difficult due to the complex interactions involved, and they suggest that immunoglobulin adsorption on solid surfaces takes place with a rather low experimental reproducibility.

Perhaps one of the most striking features that crystallographic studies have revealed is that of molecular flexibility. This kind of flexibility is expected to facilitate the formation of antibody-antigen complexes. The Fab and Fc frag ments are relatively compact; however, the whole IgG molecule is not compact (its scattering curves are anomalous and the radii of gyration of the whole molecule are larger than expected for overall close packing of regions). This segmental flexibility could explain why the dimensions of immunoglobulin G vary, and why the distance between binding sites of an antibody on an elongated molecule is 12 nm (crystalline state) but molecules can expand to reach 25 nm (end-to-end solution distance) [29-33]. This segmental flexibility might explain the poor agreement between the IgG adsorption data obtained by different authors. The IgG1, IgG2, IgG3, and IgG4 subclasses of human IgG contain two, four, five, and two disulfide bridges, respectively, between heavy chains, whereas mouse IgG1, IgG2a, and IgG2b contains three bridges each and guinea pig IgG2 also contains three [33]. Hence, the flexibility of these IgG molecules (Y- or T-shaped molecules) would be different as would their dimensions. Also, the area per molecule depends on the configuration of the IgG at the solid-liquid interface: the projected area in an end-on configuration is 20 nm2, whereas side-on is 103 nm2 [34]. A monolayer of side-on IgG is reported to correspond to an adsorbed amount of about 3 mg m-2, while a monolayer of end-on IgG corresponds to approximately 15 mg m-2 [35].

IgG adsorption is usually an irreversible process; there is practically no desorption of antibodies by dilution of IgG-coated polymer particles when they are diluted at pH 7, as can be seen in Fig. 2 [36]. Thus, although adsorption isotherms from solution appear to be of the Langmuir type, it is not possible to determine equilibrium thermodynamics binding constants from this kind of experiment. Adsorption isotherms of IgG on polymer supports usually developed well-defined plateaus that were in the range of those calculated for a close-packed monolayer of IgG molecules [28,36-43]. The results obtained with the adsorption of a monoclonal antibody (MAb) (IgG1 isotope directed against hepatitis B antigen, HBsAg) on cationic and anionic polystyrene latex particles are shown in Fig. 3. Even when the protein has the same charge sign as its adsorbent, adsorption occurs spontaneously. These results constitute an example of well-defined plateaus [37]. It should be noted, however, that step-like adsorption isotherms [27] and others without a clear plateau value [44] have also been reported. These discrepancies stress the necessity for proper characterization of both IgG and polymer supports used in IgG adsorption studies.

The conformational stability of a protein is mainly determined by intramolecular factors and solvent interactions (hydration of interfacial groups). Nevertheless, solubility is determined primarily by intermolecular effects (proteinprotein interactions), but protein molecules are solvated, so that hydration effects are also involved in changes in solubility. The energy of the hydration interaction will depend on the groups placed in the interfacial zone of the protein, and then solubility and conformational stability are closely related. Solubility is a good index of denaturation and undergoes a minimum in the neighbor-

FIG. 2 Desorption of IgG from cationic polystyrene (PS) latex by dilution at pH 7.2, 2 mM ionic strength and (20 ± 1)°C. Adsorption values (•) and final values (♦).

hood of the isoelectric pH. From the adsorption point of view the solubility of a protein is of major importance, as the method for determining adsorbed protein amount is based on the difference between the initial and the supernatant concentration. If protein molecules denature in the process, they form aggregates and precipitate in the centrifugation step. This amount of protein should be quantified as adsorbed, and it could be a cause of error [37].

Several investigators [28,36-38,40,42,43] have observed a maximum in the amount of IgG adsorbed with pH, and indicate that it is due to the decrease in conformational stability of the IgGs with increasing net charge on the molecule. This results in a greater tendency for structural rearrangements of the adsorbing molecules that create a larger surface area per molecule and cause a small amount of IgG to be adsorbed. Furthermore, at pH values away from the isoelec-tric point of the IgG, there is an increased electrostatic repulsion between adsorbed molecules that leads to a smaller amount of adsorbed IgG. Maximal protein adsorption around the isoelectric point (IEP) has been reported for IgG [28,37,38]. Figure 4 shows the adsorption plateau values of the above MAb on cationic and anionic polystyrene latex beads as a function of pH, and we can

FIG. 3 Adsorption isotherms of IgG on cationic (▲) and anionic (•) PS latex at pH 5.5, ionic strength 2 mM and (20 ± 1)°C.

FIG. 3 Adsorption isotherms of IgG on cationic (▲) and anionic (•) PS latex at pH 5.5, ionic strength 2 mM and (20 ± 1)°C.

see that maximal values occur in the neighborhood of the IEP of the protein [36]. Nevertheless, some authors have shown that the maximum appears in the IEP of the immunoglobulin-carrier complex [38].

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