The Purification of Antibody to PR8 Influenza A Virus

Abstract

The physico-chemical procedures at present available for the separation of proteins appear to possess inadequate resolving power for the isolation of specific antibodies from mixtures such as the γ-globulin fraction of human plasma [23, 3, 14]. The failure to achieve significant purification by these means is due presumably to the similarity of the various antibodies in solubility, size, shape, net charge and other physical chemical properties. A different approach takes advantage of the natural interaction of an antibody with its homologous antigen to separate the specific antibody from the mixture [8, 9, 26]. The complex is then isolated and dissociated. The recovery of antibody is facilitated by linking the antigen to a solid phase [2, 13]. Cellulose or ion exchange resins, which offer several means of forming insoluble complexes with the antigen, have proved particularly useful. The difficulty remains, however, of achieving satisfactory dissociation of antibodies from protein antigens. In such cases good yields of antibody can be obtained only by use of extremely acid or alkaline reactions. The present study has been directed primarily at overcoming this difficulty. Working on the hypothesis that the formation of the anti-gen-antibody complex results in part from electrostatic interaction between the molecules, the dissociating action of a synthetic polyelectrolyte has been studied. Polymethacrylic acid for instance has been shown to interact with proteins depending on the relative charges of the reactants [16, 21, 27]. Since PR8 influenza A virus and γ-globulin (containing the homologous antibody) bear opposite net charges between pH 5.4 and 6.5 (interisoelectric range) the possibility of a selective interaction of polymethacrylic acid with either antigen or antibody was investigated. Experiments were first performed to study the solubility of the total γ-globulin fraction (antibody) and PR8 influenza A virus (antigen) in the presence of polymethacrylic acid (PMA). Various procedures were then carried out to isolate the soluble virus antibody complex which was to be fractionated with PMA. The method was compared with the purification of antibody by means of antigens linked to ion exchange resins [13]. Although the PR8 influenza A virus and its homologous antibody comprised the test system, it is anticipated that this procedure should prove applicable to the purification of other antibodies, when a relatively pure antigen is available.