Adsorption Of Gamma-globulin Research Articles

Gamma Globulins Adsorption on Carbofunctional Polysiloxane Microspheres

Polysiloxane microspheres containing a large number of silanol groups were functionalized by reactions of these groups with organic alkoxy or chlorosilanes having chloro, amine or imidazole functions in their organic parts. The obtained imidazole groups on the microspheres were ionized by the reaction with n-octyl iodide and with methyl iodide while the chloro functions were used for quaternization of a tertiary amine. Adsorption of gamma globulins on these functionalized microspheres was studied in aqueous suspension at pH 5.2, 7.4 and 9.2. Hydrophilic–hydrophobic properties of these microspheres were examined by measuring adsorption of a hydrophobic dye, Rose Bengal. The functionalized microspheres showed higher adsorption of globulins and higher hydrophobicity than the not functionalized ones rich in silanol groups. In the case of microspheres with ionic functional groups the electrostatic forces also contribute to attractive interactions between proteins and microspheres.

Mass Transport of Macromolecules in Solution to Surfaces

Macromolecular mass transport to surfaces was measured in these studies. A method is described that was used to determine the effective diffusivity of macromolecules in sheared aqueous solutions. The adsorption of gamma globulin and albumin (γG and A, which had been covalently bound to fluorescein isothiocyanate (FITC)) onto glass planar surfaces was measured under diffusion-limited transport conditions using total internal reflection extrinsic fluorescence. The adsorbed concentration of tritium-labeled protein-FITC conjugate on the glass surface was determined by scintillation counting and compared to the fluorescence intensity measured to calibrate the experimental system. The rate of adsorption was found experimentally, and based on a Leveque type flow model a Brownian diffusivity for the adsorbing macromolecule was determined explicitly. Diffusivity was determined in isotonic phosphate-buffered saline solution (pH 7.4) for wall shear rates from 200 to 1,400 s-1. As expected, results obtained for the sheared homogeneous solutions did not appear to be a function of shear rate and were in good agreement with the literature values for Brownian diffusivity of γG and A. This approach was then extended as described in the accompanying paper to measurement of mass transport of macromolecules in sheared suspensions to surfaces.

Adsorption of γ‐globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.

Modification of polyetherurethane for biomedical application by radiation induced grafting. II. Water sorption, surface properties, and protein adsorption of grafted films.

A series of polyetherurethane films grafted by means of gamma radiation with hydrophilic or reactive monomers (2-hydroxyethyl methacrylate, 2,3-epoxypropyl methacrylate, 2,3-dihydroxypropyl methacrylate, and acrylamide) and partially chemically modified were subjected to various physico-chemical investigation methods involving water sorption, contact angle, and protein adsorption measurements. From contact angle data the interfacial free energy gamma sw between grafted films and water was calculated. It was found that the water uptake of grafted films increases with grafting yield or, in the case of grafted and afterwards chemically modified films, with reaction yield; the diffusion coefficient of water in the modified films also increases with grafting yield. Contact angle studies revealed all grafted films to have surfaces more hydrophilic than the ungrafted trunk polymer. The degree of hydrophilicity--especially of HEMA-grafted films--strongly depends on grafting conditions. For some grafted samples with high surface hydrophilicity very low interfacial free energies approaching zero were measured. The study of the competitive adsorption of bovine serum albumin, gamma-globulin, and fibrinogen from a synthetic protein solution onto modified films showed that the adsorption of albumin increases markedly with increasing grafting yields, whereas the fibrinogen and gamma-globulin adsorption only slightly increases. A correlation between interfacial free energy and protein adsorption in the sense of the "minimum interfacial free energy hypothesis" was found only for samples with grafting yields below 5%. At higher grafting yields the increased surface area complicates the analysis.

Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption

Total internal reflection fluorescence (TIRF) is a powerful new method for probing protein adsorption at solid-liquid interfaces. A quantitation method for determining the amount of adsorbed protein on hydrophilic glass and quartz surfaces has been developed. This method utilizes fluorescent, non-adsorbing dextran as a calibration molecule which approximates the diffusive properties of the protein under study. Through the use of conventional bulk solution fluorescence of both protein and calibration molecules in conjunction with a TIRF calibration, graphical quantitation of the surface concentration of adsorbed protein can be performed. quantitative results can be obtained either via a graphical procedure using fluorescence calibration standards or by a numerical method which accounts for the characteristics of the decaying evanescent, interfacial wave. The graphical quantitation procedure is outlined and demonstrated with data from five gamma-globulin adsorption experiments on hydrophilic glass and quartz surfaces. The results generated by this quantitation procedure are similar to values derived from the numerical quantitation calculation.

Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption

Total internal reflection fluorescence (TIRF) is a powerful new method for probing protein adsorption at solid-liquid interfaces. A quantitation method for determining the amount of adsorbed protein on hydrophilic glass and quartz surfaces has been developed. This method utilizes fluorescent, non-adsorbing dextran as a calibration molecule which approximates the diffusive properties of the protein under study. Through the use of conventional bulk solution fluorescence of both protein and calibration molecules in conjunction with a TIRF calibration, graphical quantitation of the surface concentration of adsorbed protein can be performed. quantitative results can be obtained either via a graphical procedure using fluorescence calibration standards or by a numerical method which accounts for the characteristics of the decaying evanescent, interfacial wave. The graphical quantitation procedure is outlined and demonstrated with data from five gamma-globulin adsorption experiments on hydrophilic glass and quartz surfaces. The results generated by this quantitation procedure are similar to values derived from the numerical quantitation calculation.

Rheumatoid factor in sera of dermatitis herpetiformis patients

Serum specimens from 44 patients with dermatitis herpetiformis (DH) and 42 control patients were analyzed with solid-phase radioimmunoassay (RIA) developed to detect IgG and IgM class antibodies against herpes simplex virus (HSV) envelope antigen. They were also tested with different tests to detect rheumatoid factor (RF) as well as serum antibodies to gastric parietal cells, glomerular basement membrane, smooth muscle, mitochondria and nuclei. Total serum IgG, IgM and IgA quantification was performed concurrently. IgM class antibodies reacting in HSV envelope antigen RIA were found in 47.8% of the DH patients, but in none of the controls. The age distribution of the IgM positive patients was in accordance with the appearance of RF, representing elderly persons. The presence of RF in serum specimens was confirmed by removing the IgM reactivity with heat aggregated human gamma globulin adsorption and in 13 patients with positive serological tests for RF. No correlation was found with the appearance of RF and duration or severity of the disease or with the detection of RF and serum total IgM concentration. In DH patients antibodies to gastric parietal cells were found in 6 and to smooth muscle in 1 of 32 specimens tested.