Aggregation of HSA, IgG, and Fibrinogen on Methylated Silicon Surfaces.

Ellipsometry was used to quantify adsorption and tapping mode atomic force microscopy to study surface aggregation of human serum albumin (HSA), immunoglobulin G (IgG), and fibrinogen (Fib) adsorbed from aqueous solutions onto methylated silicon surfaces. After exposure to air the protein monolayers were spontaneously restructured, exposing disorganized areas with heterogeneity depending on the degree of surface methylation. The aggregation patterns also depended on some properties of the adsorbed protein (such as the number of contact points with the surface), but seemed to be almost independent of the adsorption time. The results indicate that aggregates were formed due to lateral reorganization on the adsorbed layer at the air-liquid interface during the drying process. The interpretation is that the heterogeneous structures result from a thermodynamically driven interaction between the hydrophobic surface and the similarly hydrophobic air. The main conclusion that can be extracted from this work is that fibrinogen (hydrophobic and large protein) interacts more irreversibly with the silicon surfaces than IgG, and much more so than HSA, which is less hydrophobic and smaller than fibrinogen. Copyright 1998 Academic Press.

Colloidal stability of IgG- and IgY-coated latex microspheres.

The stabilization of antibody-latex complexes at high salt concentration is an event that cannot be explained by the widespread DLVO theory. Adsorption of antibodies on polystyrene latex usually leads to a loss in colloidal stability. However, after the expected particle aggregation induced by an increase in ionic strength, an 'anomalous' restabilization occurs when the electrolyte concentration increases even more. This non-DLVO behaviour can be explained taking into account the hydration forces, which become significant in hydrophilic surfaces. This restabilization has already been observed in different protein latex complexes. In the present work, a study on the stability patterns of polystyrene particles covered independently by mammalian and chicken antibodies has been performed. This study reveals that avian antibodies present a more hydrophobic surface than that of mammalian antibodies. In addition, it has been possible to obtain some information about the molecular orientation of the adsorbed antibodies from the stability experiments. This information has been corroborated by an immunoreactivity study.

Colloidal Stability of Polymer Colloids with Different Interfacial Properties: Mechanisms

An investigation is presented into the effects of interfacial properties on the colloidal stability of monodisperse polymer colloids using a sulfonate polystyrene (PS) and a styrene-hydroxyethyl methacrylate copolymer (PSHEMA) latex. Both latexes were prepared by emulsion copolymerization in the absence of surfactant. The experimental stability of these colloidal dispersions was studied in relation to the concentration (Ce) of an indifferent electrolyte. Results are compared to current models, relating the stability ratio (W) to intermolecular forces between particles. The Hamaker constant (A) obtained from the log W-log Ce plots and using the DLVO theory is much lower than the theoretical value given by the Lifshitz theory. To achieve good agreement between the experimental and theoretical A values, different corrections to the DLVO theory were introduced. First, the modified stability factor (W) was numerically estimated taking into account the hydrodynamic effect as given by L. A. Spielman [J. Colloid Interface Sci. 33, 562 (1970)]. Next, the real size of the hydrated ions was also considered. With these modifications only, small discrepancies remained between the theoretical and experimental values of the Hamaker constant in the case of the PS latex; however, the A value obtained for the PSHEMA latex studied is rather low. As HEMA is a nonionic and polar monomer, a new correction was made to the DLVO theory: the effect of electrosteric stabilization. Both steric repulsion and the electrostatic effect are shown to play a significant role in maintaining dispersion stability for the PSHEMA latex.

Effect of Storage Time on the Immunoreactivity of IgG Physically Adsorbed or Chemically Bound to Latex Beads

The main purpose of the present work is to check whether the immunoreactivity of IgG molecules, chemically bound or adsorbed on latex, shows alteration after 6 months of storage. For this purpose, agglutination tests of sensitized carboxylated beads, measuring the immunoreactivity by turbidimetric monitoring after 5 min of immunoaggregation reaction with human C-reactive protein in a stopped-flow spectrophotometer, were repeated. The results show that while the covalently bound IgG maintains and in some cases improves on its previous immunological response, the passively adsorbed IgG shows loss of activity due to a decreased amount of adsorbed protein.

Characterization of Immunoglobulin G Bound to Latex Particles Using Surface Plasmon Resonance and Electrophoretic Mobility.

The main objective of this work was the investigation of passive adsorption and covalent coupling of a polyclonal IgG and a monoclonal preparation of IgG against HSA, to a carboxyl latex particle. The functional activity of the coupled protein was then assessed by quantitative immunoassays for the antigen. Sensitized particles, with different protein coverage, were fully characterized using a range of different technologies, including electrophoretic mobility (µe), photon correlation spectroscopy, and surface plasmon resonance (SPR). The antibody-labeled particles were studied with respect to electrokinetic behavior in pH and ionic strength titration, stability, antibody functionality, and their perfomance in immunoaggregation reactions. Important differences were observed between the two sets of particle preparations throughout the series of experiments. The differences could be attributed to the coupling of the IgG molecules to the particles by the two different adsorption protocols. When proteins were chemically bound to the polymer surface it was necessary to activate the carboxyl groups with a carbodiimide (CDI) moiety that in our case was positively charged. The differences in characteristics between the adsorbed and the coupled antibody particles are thought to be due to the fact that in the covalent coupling protocol some CDI molecules remained linked to the particles, which altered the average electrical state of the outer layer in comparison with those samples where antibodies were physically adsorbed. On the other hand, the isoelectric point of the monoclonal antibody was lower (5.4 +/- 0.1) than the pI of the polyclonal antisera (6.9 +/- 0.9), which could explain why the IgG-latex complexes created with monoclonal molecules were colloidally more stable at neutral pH than those created with the polyclonal antisera. However, no immunoaggregation of antibody particles by the presence of antigen was found with the former. The use of SPR demonstrated that the equilibrium constants for the antibody-antigen recognition of the two antibody preparations were quite similar (KA polyclonal IgG = 2.8 10(8) M-1; KA monoclonal IgG = 9.5 10(7) M-1). These observations suggest that the lack of aggregation mediated by antigen demonstrated by the monoclonal antibody coupled to the latex particles may be due to this protein recognizing only one epitope in the HSA molecule. However, as the repulsive charge between antibody-latex particles counteracts the attractive forces between the antigen epitope and the antibody paratope, the greatest immunoaggregation was obtained when using latex particle-antibody complex with a low charge density (N) in the external layer. Copyright 1998 Academic Press.