Synthesis and Reactions of Hydrophilic Functional Microspheres for Immunological Studies

There is a need for hydrophilic polymeric microspheres with functional groups on their surface which can be reacted efficiently with proteins. These microspheres with antibodies (immunoglobulins) covalently bound to their surfaces constitute valuable immunoreagents capable of marking specific receptors (antigens) on cell surface membranes. The main requirements of the microspheres for the above applications are: insolubility in aqueous or organic media, absence of aggregation and of nonspecific interaction with cells and presence of suitable functional groups for covalent binding with antibodies. Hydrophobic polystyrene or poly(methyl methacrylate) latices do not meet these requirements. Copolymerization of hydrophilic monomers under suitable experimental conditions yielded microspheres with the required characteristics. Emulsion polymerization and ionizing radiation were found to constitute convenient techniques for the synthesis of hydrophilic and crosslinked (and therefore insoluble) functional microspheres ranging in diameter from 0.01 to 8 μm. By choosing suitable comonomers, it was possible to incorporate hydroxyl, carboxyl, amido, and dimethylamino functional groups into the particles. Copolymerization with isomeric vinylpyridines or dimethylamino methacrylate yielded weakly or strongly basic groups, respectively, capable of binding with acids. The experimental conditions suitable for obtaining desired particle sizes, in a relatively narrow distribution, were determined. It was found that the particle size depended to a large extent on the water solubility of the monomers, the presence or absence of stabilizer, the concentration of a surfactant, and the monomer concentration. The preferred technique to bind antibodies to the microsphere surface consisted of reacting amino groups with glutaraldehyde followed by the reaction with proteins. The use of polyglutaraldehyde instead of glutaraldehyde was also investigated. For this purpose the rate of polymerization of glutaraldehyde as a function of concentration and pH was first studied, followed by a study of the reactivity of polyglutaraldehyde micro-spheres with immunoglobulins. A recent new development of importance for cell separation is the synthesis of functional microspheres containing magnetic iron oxide. Preliminary investigations show that red blood cells and lymphocytes labeled with magnetic immunomicrospheres can be efficiently separated by means of permanent magnet. Separation of labeled from unlabeled human red blood cells was also achieved by means of a free-flow electrophoretic instrument.