Competitive adsorption of albumin and monoclonal immuno upsilon globulin molecules on polystyrene surfaces

<p>The subject of this thesis is proteins at interfaces. The main purpose of the work was to acquire more insight into the mechanism of adsorption of Bovine Serum Albumin (BSA) and monoclonal Immuno gamma Globulins (IgG's). both individually and in competition. Another aim was to achieve optimum conditions for the colloidal stability of polystyrene (PS) latices coated with these proteins, a feature that is important In the development of diagnostic test systems.<p>First, in <strong>chapter 2</strong> . a literature overview is given with special emphasis on (1) the adsorption of protein mixtures and (ii) the dynamic aspects of protein adsorption. The reason for studying the latter is that there are many indications that protein adsorption is to a certain extent irreversible. Hence, the ultimate properties of a protein adsorbate may partly be determined by its history. The structure stability of a protein molecule is considered to be one of the most Important parameters influencing its (competitive) adsorption behaviour. The heterogeneity of an adsorbed protein layer can also be related to this intrinsic property of the protein. The 'reversibility' aspects are reflected in the extents of desorption, exchange and/or displacement. The molecules that arrive the latest on the surface will be more easily removed compared to those adsorbing in a more initial stage, having more time to adapt themselves optimally.<p>In <strong>chapter 3</strong> single adsorption of BSA and monoclonal IgG's on differently charged PS latices is described. Monoclonal IgG's having different isoelectric: points were used, allowing us to systematically investigate the electrostatic aspects of Interaction. Much attention is paid to the proper purification and characterization of the materials used. The main finding in this chapter is the occurrence of maximum protein adsorption as a function of pH around the isoelectricity of the protein-latex complex, rather than that of the solute protein. An important aspect in the overall protein adsorption process is the involvement of low molecular weight ions.<p>The competition between BSA and monoclonal IgG's has been studied by sequential ( <strong>chapter 4</strong> ) and simultaneous ( <strong>chapter 5</strong> ) adsorption.<br/>IgG molecules are hardly able to displace pre-adsorbed BSA from PS latices, whereas BSA molecules are capable to partially displace pre-adorbed IgG from these surfaces. More precisely, partial displacement of IgG occurs from a positively charged PS latex at pH≈7 and from negatively charged latices at lower pH. The lower structural stability of BSA molecules compared to that of the (larger) IgG molecules may account for this effect. In addition, the extent of displacement depends on the electrostatic interaction between the respective proteins and the surface. Additional adsorption of different IgG's on BSA-coated latices follows an electrostatically determined sequence, whereas no such trend is found for the reverse case of additional adsorption of BSA on different IgG pre-coated latices. Under electrostatically attractive conditions between the secondly supplied and pre-adsorbed protein, large additionally adsorbed amounts are found, a phenomenon that may partly be attributed to the formation of a protein-protein complex on the surface.<p>Simultaneous adsorption of BSA and IgG only leads to preferential adsorption of one of the two proteins, when the proteins and the adsorbent have the same charge sign. Initially, protein molecules having the lower electrostatical affinity for the surface are retarded. Additional adsorption of this protein does occur with time, but the preference is already determined in the early stage. Fixation of the initial surface composition is also found under electrostatically attractive conditions between proteins and adsorbent. Furthermore, competitive adsorption between different IgG's is governed by electrostatic interactions. It is concluded that electrostatic interactions are important in competitive protein adsorption. The finding is helpful to predict the ultimate preference in the adsorbate.<p>In <strong>chapter 6</strong> kinetic aspects of the adsorption on macroscopic PS surfaces is studied by reflectometry and streaming potential measurements. The initial adsorption rate and plateau adsorption of BSA both show a maximum around its isoelectric point, leading to the conclusion that the final surface composition is already frozen in in the initial stages. For one monoclonal (isoelectric point ≈7.0), the initial adsorption rate was found independent of the pH. Apparently, other than electrostatic interactions are dominating. Finally, single and competitive adsorption of these proteins are comparable with those observed on the latices