Sedimentation FFF is a sensitive method for measuring the mass of an adsorbed layer on colloidal particles and it is able to evaluate surface concentrations of immunoglobulins or antibodies (Igs) adsorbed to polystyrene latex particles [1-2]. Immunoglobulin A (IgA) is the main immunoglobulin in mucous secretions, including tears, saliva and colostrum. IgA believed to play an important role in defense mechanisms against infections and because it is resistant to degradation by enzymes, secretory IgA provides protection against microbes proliferating in body secretions, especially those of the digestive and respiratory tracts [3]. Binding of Igs onto a hydrophilic surfaces lies at the center of a multitude of biological, biomedical and biotechnological application. Chromatographic supports, membranes and latex particles are only few examples of substrates on which immunoproteins may be adsorbed. Immobilization of the Igs may be due either by physical adsorption or by covalent binding to functionalized surface groups. Appropriate conditions for binding are as varied as the type of molecules concerned and their chemical binding properties. IgA show a strong tendency to adsorb on polystyrene (PS), under several different chemical-physical conditions. In this work the mass of IgA adsorbed onto PS microsphere standard has been determined from SdFFF fractograms obtained by varying the mobile phase composition. The compatibility of the SdFFF channel with several buffers used as carriers was proven, in particular, density, chemical composition, its pH and its ionic content, have been considered as experimental variables. Their ability to correctly fractionate the PS and the PS-IgA complex has been tested on two monodispersed polystyrene latex microbeads samples. The surface reactivity of the complex PS-IgA has been verified by preparing the ternary complex PS-IgA-antiIgA (specific antigen-antibody reaction) and by monitoring the increase of mass by SdFFF. The negative control was the reaction between the complex PS-IgA and anti IgE, which did not occurred as proven by the registered fractograms.
Sedimentation Field-Flow Fractionation of polystyrene beads coated with IgA: carrier composition effects on complex characterization.
BREGOLA, Letizia;CONTADO, Catia;DONDI, Francesco
2007
Abstract
Sedimentation FFF is a sensitive method for measuring the mass of an adsorbed layer on colloidal particles and it is able to evaluate surface concentrations of immunoglobulins or antibodies (Igs) adsorbed to polystyrene latex particles [1-2]. Immunoglobulin A (IgA) is the main immunoglobulin in mucous secretions, including tears, saliva and colostrum. IgA believed to play an important role in defense mechanisms against infections and because it is resistant to degradation by enzymes, secretory IgA provides protection against microbes proliferating in body secretions, especially those of the digestive and respiratory tracts [3]. Binding of Igs onto a hydrophilic surfaces lies at the center of a multitude of biological, biomedical and biotechnological application. Chromatographic supports, membranes and latex particles are only few examples of substrates on which immunoproteins may be adsorbed. Immobilization of the Igs may be due either by physical adsorption or by covalent binding to functionalized surface groups. Appropriate conditions for binding are as varied as the type of molecules concerned and their chemical binding properties. IgA show a strong tendency to adsorb on polystyrene (PS), under several different chemical-physical conditions. In this work the mass of IgA adsorbed onto PS microsphere standard has been determined from SdFFF fractograms obtained by varying the mobile phase composition. The compatibility of the SdFFF channel with several buffers used as carriers was proven, in particular, density, chemical composition, its pH and its ionic content, have been considered as experimental variables. Their ability to correctly fractionate the PS and the PS-IgA complex has been tested on two monodispersed polystyrene latex microbeads samples. The surface reactivity of the complex PS-IgA has been verified by preparing the ternary complex PS-IgA-antiIgA (specific antigen-antibody reaction) and by monitoring the increase of mass by SdFFF. The negative control was the reaction between the complex PS-IgA and anti IgE, which did not occurred as proven by the registered fractograms.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
