Studies on the purification of antibodies

Abstract

It has been known for years that when precipitates are produced in serum by salts of the heavy metals, immune bodies present are more or less completely precipitated. Whether the immune bodies are precipitated by the metal directly or whether merely adsorbed by the rather voluminous protein precipitate formed has never been determined. It seemed a possibility that the metal might actually enter into combination with the immune body, and that by repeated precipitation of such a combination, purification of the immune body might be brought about. In preliminary experiments with anti-sheep hemolysin and typhoid agglutinin, using the chlorides or sulfates of copper, nickel, zinc, mercury and lead as precipitants, it was found that a good deal of these antibodies could be dissolved out of the precipitates. On account of the large amount of protein present, however, it was found that any really effectual purification of the antibody was impossible.

It was determined therefore, to try the effect of metallic salts on antibody already partially freed of protein by the method of specific absorption followed by subsequent dissociation from appropriate cellular antigens. This we have done both with antisheep hemolysin and with typhoid agglutinin. But on account of the interference of the metal salts with the action of complement in hemolysin experiments, the work has been more successful with bacterial agglutinin.

With typhoid agglutinin the preliminary purification was brought about by first sensitizing large amounts of formol-killed typhoid bacilli as nearly as possible to saturation with agglutinin, then washing the sensitized bacteria free of serum in repeated changes of saline solution, and then dissociating the agglutinin from the bacteria either with water at 55° C for one half to one hour, or with N/200 NaOH at room temperature for five minutes.