Protein A-immobilized microporous polyhydroxyethylmethacrylate affinity membranes for selective sorption of human-immunoglobulin-G from human plasma.
Microporous membranes made of poly(2-hydroxyethylmethacrylate) [poly(HEMA)] carrying protein A were used for selective sorption of human-IgG from human plasma. Poly(HEMA) membranes were prepared by a photo-polymerization technique, and activated by cyanogen bromide (CNBr) in an alkaline medium (pH 11.5). Bioligand protein A was then immobilized by covalent binding onto these CNBr-activated membranes. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The non-specific adsorption of protein A on the plain poly(HEMA) membranes was 2.9 microg cm(-2). Maximum protein A immobilization was observed at pH 9.5. Up to 186 microg cm(-2) was immobilized on the CNBr-activated poly(HEMA) membranes. The maximum adsorption of human-IgG on the protein A-immobilized poly(HEMA) membranes was observed at pH 8.0. The non-specific adsorption of human-IgG onto the plain poly(HEMA) membranes was low (about 4.4 microg cm(-2)). Higher human-IgG adsorption values (up to 394 microg cm(-2)) were obtained in which the protein A-immobilized poly(HEMA) membranes were used. Much higher amounts of human-IgG (up to 489 microg cm(-2)) were adsorbed from human plasma. Up to 91% of the adsorbed human-IgG was desorbed by using 0.1 M aminoacetic acid as elution agent. The adsorption-desorption cycle was repeated ten times using the same polymeric membranes. There was no remarkable reduction in the adsorption capacity of the protein A-immobilized poly(HEMA) membranes.
Denizli
A
A
Hacettepe University, Chemistry Department, Ankara, Turkey. denizli@eti.cc.hun.edu.tr
Arica
Y
Y
D003488:Cyanogen Bromide / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000097:blood / Q000378:metabolism*; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D010949:Plasma / Q000378:metabolism*; D011102:Polyhydroxyethyl Methacrylate / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000378:metabolism*
D007074:Immunoglobulin G; D008567:Membranes, Artificial; D013205:Staphylococcal Protein A; D011102:Polyhydroxyethyl Methacrylate; D003488:Cyanogen Bromide