Preparation of superparamagnetic immunomicrospheres and application for antibody purification

A novel and effective protocol for the preparation of superparamagnetic immunomicrospheres has been developed. First, micro-size magnetic poly (methacrylate-divinylbenzene) (PMA-DVB) spheres were prepared by a modified suspension polymerization method. The oleic acid coated magnetite (Fe₃O₄) nanoparticles made by coprecipitation were mixed with monomers of MA, DVB, and initiator benzoyl peroxide (BPO) to form oil in water emulsion droplets with the presence of poly (vinyl alcohol) (PVA-1788) as a stabilizer. The polymerization reaction was carried out in a 2-L beaker equipped with four vertical stainless steel baffleplates by increasing the temperature of the mixture at a controlled rate. The resulting magnetic microspheres are micro-sized (less than 8μm in diameter) and 80 percent of them are in the size ranging from 1 to 5 μm. Then, they were highly functionalized via ammonolysis reaction with ethylenediamine, and the surface amino-modified magnetic microspheres were obtained. The morphology and properties of these magnetic microspheres were examined by SEM, TEM, VSM, and FT-IR. Affinity ligand protein A (ProtA) was covalently immobilized to the amino-modified magnetic microspheres by the glutaraldehyde method. These ProtA-immobilized magnetic immunomicrospheres were effective for affinity bioseparation processes, as was demonstrated by the efficient immunoaffinity purification of antibodies IgG2a (22mg per gram of microspheres) from mouse ascites. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 2205–2211, 2004