Immunoglobulins and their fragments on solid surfaces

J.A.G. Buijs

Research output: Thesisinternal PhD, WU


<strong>Summary</strong><p>Adsorption of immunoglobulin G (IgG) is a common step in the production of immunological tests and biosensors. The use of IgG in these applications stems from its ability to specifically bind all kinds of molecules (antigens). In these tests the IgG molecules are immobilised onto solid surfaces, which is necessary to make visible the IgG-antigen binding. Upon adsorption, however, the IgG molecules may lose their ability to bind antigens. If the antigen binding sites are located close to the sorbent surface, this could reduce the accessibility for antigens. A second problem which may occur upon adsorption is related to structural rearrangements of the protein which could also cause a reduction in the antigen binding capacity.<p>The purpose of the present work was to acquire more insight into the role of the various interactions between IgG molecules and sorbent surfaces on the adsorption behaviour and, especially, how these interactions determine the adsorbed state of the IgG molecules.<p>After a general introduction in Chapter 1, in Chapter 2 an overview is given of the physical properties and some general aspects of the adsorption behaviour of IgG.<p>The different types of protein-sorbent interactions are systematically studied by performing experiments under various system conditions. The influence of the sorbent surface hydrophobicity is investigated using both a hydrophobic and a hydrophilic surface. Chapter 3 describes the adsorption of the proteins onto three polymer latices. Two of these latices are hydrophobic, one being positively and the other negatively charged and the third latex is negatively charged and hydrophilic. The adsorption experiments described in Chapters 4, 5, and 6 are performed using a hydrophilic silica and a hydrophobic methylated surface. Electrostatic interactions are varied by choosing various values for the pH and ionic strength. The experiments are performed using two monoclonal IgGs directed against the pregnancy hormone human chorionic gonadotropin (hCG). These two monoclonals differ in isoelectric point (i.e.p.). As the orientation of the adsorbed IgG molecules is expected to be influenced by differences in adsorption behaviour of the single domains, experiments are also performed using the fragments, i.e., the F(ab') <sub><font size="-1">2</font></sub> and Fc fragments, of the whole IgG molecule.<p>Chapter 3 focuses on the equilibrium adsorption of the two IgGs and their corresponding F(ab') <sub><font size="-1">2</font></sub> fragments on polymeric surfaces. Special attention is paid to the electrokinetic properties of the protein/sorbent complexes. Furthermore, the obtained adsorbed amounts at saturation conditions are correlated to the possible orientations of the proteins in the adsorbed state. The proteins show a high affinity for the hydrophobic latices and this affinity is barely influenced by the electrostatic interactions involved. However, at saturation level the adsorbed amount depends on the overall electrostatic interactions resulting in a maximum when the protein charge is to a large extent compensated for by the sorbent surface charge. For the hydrophilic latex, adsorption is absent when the proteins are electrostatically repelled. At hydrophobic polystyrene surfaces, the proteins adsorb mainly in an end-on orientation. In contrast, in the case of a negatively charged hydrophilic: latex and high cationic charge densities on the protein, the adsorbed amounts correlate with a closely-packed monolayer of side-on oriented proteins. The trends in the adsorption behaviour are similar for IgG and its corresponding F(ab') <sub><font size="-1">2</font></sub> fragment. Nevertheless, there is some evidence that IgG adsorption is more strongly driven by interactions other than electrostatic ones, hence, by hydrophobic interactions and/or conformational changes.<p>Dynamic aspects of the adsorption process of the two IgGs and their F(ab') <sub><font size="-1">2</font></sub> and Fc fragments are described in Chapter 4. This information is obtained by monitoring the adsorption process in real-time using reflectometry. Under all conditions, the two IgGs and their F(ab') <sub><font size="-1">2</font></sub> and Fc fragments adsorb onto the silica and methylated surfaces. However, the adsorption rate of IgG and F(ab') <sub><font size="-1">2</font></sub> on a hydrophilic silica surface is retarded when the proteins are electrostatically repelled by the sorbent surface. This effect is stronger for F(ab') <sub><font size="-1">2</font></sub> fragments than for the whole IgG molecule, whereas the adsorption rate of Fc is not significantly affected. This observation shows that there must be an additional driving force for adsorption of the Fc fragments compared to that of the F(ab') <sub><font size="-1">2</font></sub> fragments. On the methylated surface, hydrophobic interactions largely compensate for the retardation of the initial adsorption rate. Both IgGs show a maximum saturation adsorption around their i.e.p. values and these maxima are less pronounced at higher ionic strength. The decrease in adsorbed amounts when the pH is shifted from the i.e.p. of the proteins is probably caused by the formation of a more expanded structure of the protein owing to a rise of its net charge density. Desorption of the proteins after replacement of the protein solution by the pure buffer was determined after fifteen minutes. The desorbed amounts indicate that the proteins are more tightly bound to methylated surfaces than to silica. Furthermore, a relatively large fraction of IgG and Fc desorbs from silica at low ionic strength around their respective i.e.p. values, whereas this phenomenon is not observed for F(ab') <sub><font size="-1">2</font></sub> . This indicates that under these experimental conditions a fraction of the Fc fragments forms rather weak bonds with the sorbent surface, which are easily broken upon dilution.<p>A quantitative relationship between the adsorption and the secondary structure of the proteins is described in Chapter 5. This information is attained by measuring Fourier Transformed Infrared spectra of adsorbed IgG and F(ab') <sub><font size="-1">2</font></sub> layers. The obtained infrared spectra consist of two absorption bands, the amide I and amide II regions. The amide II region is rather insensitive to the structure of the adsorbed proteins and is related to the adsorbed amounts. The amide I region is sensitive to the secondary structure and is therefore used for the evaluation of this structure. The results show that the amounts of adsorbed IgG decrease with an increasing net charge density on the protein. This decrease is accompanied by a decrease in β-sheet structure which suggests that IgG adsorbs in a less compact conformation. The adsorption-induced reduction in the β-sheet content is larger at hydrophobic methylated surfaces than at hydrophilic silica surfaces. At higher ionic strength, the distribution of the amino acid residues over the structural components depends less on the pH. This feature is reflected by a smaller sensitivity of the adsorbed amount on the pH variation. The F(ab') <sub><font size="-1">2</font></sub> fragments contain a higher fraction of β-sheet than IgG and these fractions are less influenced by adsorption. Therefore, it is concluded that the F(ab') <sub><font size="-1">2</font></sub> fragments have a higher structural stability than whole IgG molecules.<p>In Chapter 6, the adsorption behaviour of the two IgGs and their F(ab') <sub><font size="-1">2</font></sub> fragments together with the binding of antigen hCG to the adsorbed proteins, is studied using reflectometry. From Chapter 5 it was inferred that the Fc part of an IgG molecule is more flexible than the Fab part. The higher flexibility promotes adsorption of the Fc part, which implies that the Fab parts are directed towards the solution. This is reflected in higher antigen binding ratios under conditions in which the adsorption of F(ab') <sub><font size="-1">2</font></sub> fragments is unfavourable. Furthermore, it is observed that the orientation of an adsorbed IgG molecule with an uneven charge distribution is strongly influenced by electrostatic interactions. When the Fab parts are electrostatically attracted by the sorbent surface, this may even result in a total absence of the antigen binding capacity.<p><strong>Perspectives</strong><p>In the present study, we obtained a wealth of information about the interactions involved in IgG adsorption onto solid sorbent surfaces. For practical applications it is desirable to use this information to control the adsorbed state of the IgG molecules. For example, in the production of immunological tests the IgG molecules will have to adsorb with their antigen binding sites accessible for the antigens and without structural changes affecting the antigen binding capacity.<p>In Chapter 5 it was observed that the F(ab') <sub><font size="-1">2</font></sub> fragments have a relatively high structural stability and that the structure of F(ab') <sub><font size="-1">2</font></sub> remains stable under a wide range of adsorption conditions. This information suggests that in the adsorbed state the F(ab') <sub><font size="-1">2</font></sub> domains retain their ability to bind antigens.<p>The orientation of the IgG molecules can strongly be influenced by varying the adsorption conditions. In Chapter 3 it is inferred that IgG adsorbs in an end-on orientation at hydrophobic polystyrene latices. For adsorption onto the silica and methylated surfaces, the adsorbed amounts indicate that at least a large fraction of the IgG molecules are adsorbed in such an orientation. In an end-on orientation the antigen binding sites could be directed towards the solution or be attached to the sorbent surface. The differences in adsorption behaviour between F(ab') <sub><font size="-1">2</font></sub> and Fc fragments can now be used to obtain an adsorbed state of the whole IgG molecule in which the antigen binding sites are preferentially directed to the solution. The present study reveals that there are two possible mechanisms for directing the orientation of the adsorbed IgG molecules. First, as shown in Chapter 6, the IgG orientation can be influenced by electrostatic interactions if the charges of the protein are unequally distributed over the Fc and F(ab') <sub><font size="-1">2</font></sub> fragments. Thus, a favourable orientation can be obtained if the Fe fragment is electrostatically attracted while the F(ab') <sub><font size="-1">2</font></sub> fragment is electrostatically repelled by the charges on the sorbent surface. Second, a difference between the structural stabilities of the Fc and F(ab') <sub><font size="-1">2</font></sub> fragments may cause different adsorption affinities of the respective fragments. As the Fc fragment is structurally less stable than the F(ab') <sub><font size="-1">2</font></sub> fragment, the adsorption of an IgG molecule by its Fc fragment is favoured.<p>However, it should be realised that these two mechanisms for directing the orientation of adsorbed IgG molecules are only effective under conditions in which the adsorption of the F(ab') <sub><font size="-1">2</font></sub> fragment is suppressed. Since the F(ab') <sub><font size="-1">2</font></sub> fragment shows a high affinity for hydrophobic surfaces, this implies that the orientation of the IgG can only be directed by electrostatic interactions if the sorbent surface is hydrophilic. Furthermore, it is observed that the adsorption of the fragments is only influenced by the differences in structural stability if the adsorption is not promoted by hydrophobic and/or electrostatic interactions.<p>In conclusion, we can state that the orientation of the adsorbed IgG molecules can be adjusted by the adsorption conditions including modification of the sorbent surface. To obtain an optimal IgG orientation, these conditions require a sorbent surface with a hydrophilic character and a controlled contribution of electrostatic interactions.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Lyklema, J., Promotor, External person
  • Norde, W., Promotor, External person
Award date22 Sep 1995
Place of PublicationS.l.
Print ISBNs9789056510152
Publication statusPublished - 1995


  • immunoglobulins
  • immunological techniques
  • elisa
  • chemical structure
  • organoleptic traits
  • biological properties

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