Quantification of adsorbed human serum albumin: A comparison between radioimmunoassay and simple null ellipsometry

Radioimmunoassay (RIA) and null ellipsometry are two common methods to quantify adsorbed proteins. However, the accuracy of null ellipsometry with a constant protein refractive index (n 1.465, k 0) at l 632.8 nm has this far not been explored. The present study compared the methods, and the degree of agreement between the simplified single wavelength null ellipsometry and RIA to quantify adsorbed proteins was explored on different surfaces. The quantification methods agreed well when A, ngstro¨m smooth hydrophilic or hydrophobic silicon surfaces, and freshly radio-labelled proteins were used. Some discrepancies were noted when either rough surfaces or stored and aged labelled proteins were used. The differences decreased when the aged protein solution was equilibrated with freshly dissolved proteins at room temperature (RT) for a few hours prior to the surface incubations. Significant differences were also noted between the methods when albumin was adsorbed at it’s iso-electric point (pH 4.8)

ELLIPSOMETRIC QUANTIFICATION OF ANTIGEN-ANTIBODY PRECIPITATES ADSORBED ON ANTIGEN-COATED SILICON SURFACES. A NEW PRINCIPLE FOR IMMUNOCHEMICAL ANALYSIS

Des précipités antigène-anticorps formés dans un agargel s'adsorbent sur des surfaces de silicium recouvertes d'un support, par exemple un antigène, qui a de l'affinité pour le précipité. La quantification des precipités adsorbés peut être déterminée, après que le gel ait été éliminé, par ellipsométrie avec une grande résolution latérale. Cette méthode a été dénommée "precipitate adsorption on surface" (PAS). Dans un système modèle comprenant de la γ-globuline, de l'albumine, ainsi que les anticorps correspondants, on a constaté que la méthode PAS est simple et très sensible pour la quantification de récepteurs biologiques, tels que les antigènes, exposés sur une surface solide. En utilisant une technique qui permet d'éliminer sélectivement les anticorps de l'antiserum contribuant au précipité, il est également possible d'étudier les relations immunochimiques entre antigènes. La résolution analytique de ce principe est beaucoup plus grande que celle des méthodes immunochimiques traditionnelles.Antigen-antibody precipitates formed in an agargel adsorb on silicon surfaces coated with a substrate, e.g. antigen, which has affinity to the precipitate. Quantification of adsorbed precipitates may be determined, after removal of the gel, by ellipsometry with high lateral resolution. The method has been called precipitate adsorption on surface (PAS). In a model system consisting of γ-globulin, albumin together with corresponding antibodies, it was noted that the PAS-method is simple and very sensitive for quantification of biological receptors, like antigens, exposed on a solid surface. With the use of a technique of selective removal of antibodies from the antiserum contributing to the precipitate, it was also possible to investigate immunochemical relationships between antigens. The analytical resolution of this principle was much higher than traditional immunochemical methods

The Elutability of Fibrinogen by Sodium Dodecyl Sulphate and Akyltrimethylammonium Bromides.

The elutability of adsorbed fibrinogen by cationic surfactants of different chain lengths (dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, cetyltrimethylammonium bromide), and an anionic surfactant (sodium dodecyl sulphate (SDS)) was studied using in situ ellipsometry. The concentrations of the surfactants were twice the CMC in water and for fibrinogen, 0.4 mg ml−1. The investigation was carried out for two model surfaces: methylated silica (hydrophobic) and silica (hydrophilic and negatively charged, at pH 7). As a complement, a surface with a gradient in surface hydrophobicity was used. The end points of the gradient are similar to the methylated silica and silica surfaces with respect to hydrophobicity. All the surfactants adsorbed on the methylated silica surfaces, whereas only the cationic surfactants adsorbed on the silica surface. The adsorption of fibrinogen was 0.64 ± 0.03 μg cm−1 and 0.35 ± 0.03 μg cm−2 on the methylated silica and silica surfaces, respectively. Addition of surfactant led to a decrease in the amount of fibrinogen adsorbed on the methylated silica surface for all the surfactants, but only SDS affected the amounts adsorbed on the silica surfaces to any great extent. Despite the fact that the cationic surfactants adsorbed onto the silica surface, they did not affect the amount of fibrinogen adsorbed. The removal of protein decreased for the alkyltrimethylammonium bromides with increasing hydrophilicity of the gradient surfaces, and the amount of fibrinogen remaining after surfactant treatment decreased slightly for SDS. The effect of the chain length of the surfactant on elutability was small. The rate of removal of fibrinogen by the surfactants was found to be slower for SDS compared with the alkyltrimethylammonium bromides at the methylated silica surface, and at the hydrophobic end and in the intermediate part of the gradient.Adsorption from mixtures of surfactant and fibrinogen was also studied and the effects of cationic and anionic surfactants were quite different. The adsorption of fibrinogen was increased in the presence of the cationic surfactants, especially on the silica surface, but decreased in the presence of SDS. As surfactant adsorption onto clean surfaces is reversible with respect to dilution it might be assumed that the adsorbate mainly consists of fibrinogen. A trend was observed for the amounts of fibrinogen remaining after rinsing with buffer; the amounts increased with decreasing length of the surfactant hydrocarbon chain

Ellipsometry and radio-labelling studies on the adsorption of human serum albumin (HSA) and anti-HSA to hydrophobic silicon

Ellipsometry and radio-labelling techniques were employed to study the deposition of human serum albumin (HSA) followed by excess of mono- or polyclonal anti-HSA to methylated silicon. Adsorbed HSA, and [HSA-monoclonal anti-HSA] layers were rapidly removed by 0.1-0.5% SDS, but [HSA-polyclonal anti-HSA] layers were not. The results suggest that antigens and polyclonal antibodies crosslink on surfaces, thereby stabilizing the protein films.Upon five repetitive and alternating exposures to excess of HSA and polyclonal anti-HSA, it was observed that the ellipsometric thickness did not increase during HSA incubations but did so during the polyclonal antibody incubations. When [HSA-polyclonal anti-HSA] multilayers were exposed to 125I-HSA, a low increased deposition with time of 125I-HSA was observed. HSA was, however, not able to remove 125I-HSA from [125I-HSA-polyclonal anti-HSA] films. The protein deposition and binding characteristics are presently not well understood but may be explained by a low HSA deposition to surface-bound polyclonal antibodies, the multiplicity of antibody binding sites on each HSA molecule, and a partial disruption of the antigen-antibody complex structure upon incubation in excess of HSA

Competition Between Fibrinogen and a Nonionic Surfactant at Adsorption to a Wettability Gradient Surface

The competition between mixtures of fibrinogen and a non-ionic surfactant (C12E5) with respect to adsorption onto a wettability gradient solid surface was studied by the use of ellipsometry. The effects of surface hydrophobicity and surfactant association were investigated. Furthermore the effect of clouding of the surfactant was studied by performing measurements at temperatures above and below the cloud point. At all concentrations, the fibrinogen (0.02–0.40 mg ml−1) was preferentially adsorbed onto the hydrophilic part of the gradient surface. At surfactant concentrations above and around the CMC, the protein was inhibited from adsorbing by the surfactant at the hydrophobic as well as in the intermediate part (50° ⩽ contact angle ⩽ 80°) of the gradient. As the surfactant concentrations was further reduced the protein was able to compete and adsorb onto the whole or parts of the gradient surface. In the case of a surfactant concentration of two-fifths of the CMC, the competitive power of the surfactant increased with temperature and the surfactant could hinder protein adsorption over a larger interval of the gradient surface. These observations were also verified by in situ measurements on non-gradient surfaces. The competition can be explained by considering the main interactions between protein and surfactant with the surface. In this respect cooperation in the self-association of the surfactant seems to be of great importance. The use of gradient surfaces makes it possible to observe subtle changes in these interactions

Peptide functionalized poly(L-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivs. (PLL-g-PEG/PEG-peptide) were assembled from aq. solns. on titanium (oxide) surfaces. The polymers were characterized by NMR in order to det. quant. the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomol. adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 DegC or 37 DegC, whereas clean, uncoated titanium surfaces adsorbed approx. 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clin. performance. [on SciFinder (R)