The Estimation of Circulating Antibody-antigen

It has generally been observed that during the course of immunization the appearance of antibody in circulation follows the disappearance of antigen (1, 2). Appreciable amounts of free antigen and its homologous free antibody apparently do not coexist in a single system (3). Existence of complexes consisting of circulating bound antibody and antigen could depend upon several factors. The earliest antibody formed during immunization may conceivably combine with antigen present in circulation. Even if the aggregates formed fail to precipitate they may escape detection by the serologic methods thus far developed. Present immunologic methods adequately detect free antibody by reactions resulting from addition of antigen, and similarly, free antigen can be detected by addition of antibody. These methods, however, would not identify antibody-antigen complex combined in dvo. It seemed worth while to learn whether bound antibody and antigen may coexist in immune serum without showing serologic activity as such, and whether any latent serologic activity of such a complex can be detected by dissociation and reassociation in vitro. Previous studies suggested possible approaches. Thrombin formed in circulation is progressively inactivated by combination with antithrombic substances to form inert thrombin-antithrombin complexes. Active thrombin can be recovered by dissociating thrombinantithrombin complex by brief alkali treatment in the cold. Addition of ethanol to the dissociated constituents prevents reassociation upon subsequent neutralization (4). A similar procedure has also been employed in the dissociation of precipitated complexes of antigen and antibody. The antigen used was coupled with acidic groups capable of reacting with alumina at an alkaline pH to form insoluble precipitates (5). This allowed recovery of dissociated antibody that was 88 to 100 per cent pure. The combining ratio of antigen with antibody was not altered. It was found that dissociation began at a pH of approximately 11 and was more pronounced the higher the pH. Electrophoretic, sedimenta-