554 BIOLOGY OF PNEUMOCOCCUS 



fraction may be removed by suspending the alcoholic precipitate 

 in water or by adding sodium chloride to effect solution, then ad- 

 justing the reaction to pH 5.0. The precipitate thus formed is 

 separated by centrifugation. The importance of maintaining an 

 even, low temperature during contact of the alcohol with serum 

 is emphasized because a slight increase in temperature was be- 

 lieved to result in solution of the precipitate and in partial de- 

 struction of antibody. A yield of at least 80 per cent of the pro- 

 tective substance originally present in the serum is obtained by 

 this method. 



Felton reported that several different lots of serum concen- 

 trated by the method had been used therapeutically without caus- 

 ing untoward reactions. Some modifications of the original tech- 

 nique have been found advisable during routine practice, chiefly 

 those directed toward avoiding denaturation of the immune globu- 

 lin. 



Other Methods: An objectionable feature of the antibody solu- 

 tions prepared by the methods of Gay and Chickering and of Hun- 

 toon is the difficulty in separating the cellular components of 

 Pneumococcus from the antigen-antibody precipitate. Felton and 

 Bailey 420 found that 0.01 molar sodium carbonate was the most 

 satisfactory of the agents tried for dissociating the polysaccha- 

 ride-antibody combination. Dissociation took place on heating the 

 specific precipitate in the presence of sodium carbonate, disodium 

 phosphate, saccharose, or sodium chloride, and the protective sub- 

 stance could then be precipitated by sodium sulfate. The released 

 antibody, however, was found to contain a substance antagonistic 

 to specific protection, thus suggesting that not all the capsular 

 polysaccharide had been removed. 



In a later study, Felton 410 reported that calcium or strontium 

 phosphate could also be used to dissociate specific carbohydrate- 

 antibody precipitate yielding a water-insoluble protein that con- 

 ferred protection on mice. Felton and Kauffmann 429 observed that 

 the zone of minimal solubility of the immune fraction isolated by 



