Isolation of Antibodies on Antigen–Polystyrene Conjugates

Abstract

THE isolation of precipitating antibodies in a purified form has been the subject of several investigations during the past few years. The property of the specific combination of antibodies with their homologous antigens has been exploited for this purpose. In principle, a general procedure for the isolation of antibodies would involve the following steps: (1) formation of insoluble antibody–antigen complexes, (2) dissociation of these complexes, and (3) separation of the purified antibodies from the antigen(s). Obviously, the last step would be facilitated if the antigen were insoluble or could be made insoluble by coupling it via stable covalent chemical bonds to a supporting medium. For example, in previous investigations protein antigens were coupled to fibrinogen¹, ion-exchange resins² and cellulose³. Since these supporting media were endowed with ion-exchange properties, it is conceivable that serum proteins may be bound to them non-specifically. In order to avoid or at least to minimize non-specific absorption of serum proteins, in the present study, protein antigens were coupled through stable azo bonds to a non-polar polystyrene (Dow ‘Styron PS-2’ obtained through the courtesy of the Physical Laboratory, Dow Chemical Co., Midland, Michigan) framework. For this purpose polystyrene was nitrated⁴. The ensuing polynitro polystyrene was then reduced to polyamino polystyrene^4,5 and the latter compound diazotized. The soluble protein antigens were then coupled to the polydiazotized polystyrene at pH 7.5. The resulting insoluble Styron–antigen conjugates are dark brown precipitates. At the beginning of the study we were not aware that a somewhat similar procedure had been used for coupling proteins to polystyrene^6,7.