Studies on the purification of antibodies. III. Certain methods for the precipitation of pneumococcus protective antibody.

Abstract

We have tried to apply the principles developed in our work on typhoid agglutinin to the purification of pneumococcus protective antibodies. The task has turned out to be particularly difficult. The great solubility of the pneumococcus makes it impossible to get the primary extracts of sensitized bacteria as free from bacterial substances as in the case of typhoid. The variability in the resistance of mice to pneumococcus infection, and the variable virulence of pneumococcus under culture, make quantitative observations impractical. For these reasons, we have been unable to get consistent results which could be repeated with regularity. Nevertheless, we did succeed many times in precipitating the pneumococcus protective body in a way which leaves no doubt that the same methods can be applied as were used in the purification of typhoid antibody.

Our primary material was the protective body obtained in alkaline solution by the method of Huntoon, or by a modification which consisted in extracting in N/500 NaOH instead of in 1/4 percent NaHCO_3. Precipitation could frequently be brought about without the addition of any metallic salt, by adding hydrochloric acid to a hydrogen ion concentraction between pH 4.0 and 4.6. The following protocol is illustrative of these results: “Isoelectric” Precipitation, and Recovery in the Dissolved Precipitate, of Pneumococcus Protective Antibody.

Ten cc. portions of an alkaline solution of pneumococcus protective antibody were brought to the indicated pH by addition of 0.15 cc. N/40 HC1, and allowed to stand in the ice box overnight. The precipitate was centrifuged, dissolved in N/400 NaOH, and placed on test. Each mouse received 0.5 cc. protective material, and 0.5 cc. 18 to 24 hours broth culture of pneumococcus Type 1, diluted 1-200, i. e., 0.025 cc. of culture. Each dilution was tested on two mice.

The addition of copper chloride, as in the case of typhoid antibody, was found to greatly facilitate precipitation of the antibody. The most favorable strength of copper chloride was about M/2200. A final concentration of M/1100 had a distinct destructive effect on the pneumococcus antibodies.