548 BIOLOGY OF PNEUMOCOCCUS 



technical manipulations were laborious, the preparations varied in 

 strength, were not stable, and frequently gave rise to severe reac- 

 tions in patients. 



Chemical methods. Chemical methods of refining antipneumococ- 

 cic serum are more practicable and have received greater recogni- 

 tion. 



Ammonium Sulfate: In the investigations made by Avery, 32 the 

 serum used was obtained from horses immunized with cultures of 

 pneumococci of Types I and II. Avery's work showed that the an- 

 tibodies in antipneumococcic serum are absent from the albumin 

 fraction but associated with the globulins, and while not associ- 

 ated wholly with pseudoglobulin, precipitation by 38 to 42 per 

 cent saturation with ammonium sulfate offered a promising 

 method for the practical purification of antipneumococcic serum. 



Avery's experiments with ammonium sulfate were confirmed by 

 the investigations of Banzhaf (1925), 70 which stimulated a con- 

 tinued interest in the method. By a modification of Avery's method, 

 and using plasma instead of serum, Brown and Knowles (1932) 153 

 attempted to increase the yield of antibody and to determine the 

 comparative protective value of the euglobulin and pseudoglobulin 

 fractions. The percentage recovery obtained is not clear, but the 

 results suggested that the bulk of protective antibody is to be 

 found in the pseudoglobulin. 



In studying the "non-antibacterial" factors in the therapeutic 

 action of immune serum on dermal pneumococcal infection in rab- 

 bits, Sabin (1933) 1206 used the ammonium sulfate method of frac- 

 tionation. A zonal effect was observed in the experiments, and, be- 

 cause of this, Sabin stated that "the 30 to 50% ammonium sulfate 

 globulin, either total or water-insoluble, would be quite undesirable 

 for therapeutic purposes." Felton and Kauffmann (1933), 429 on 

 the other hand, found that the highest protective potency for mice 

 in relation to the amount of protein was in portions precipitated 

 by ammonium sulfate between 36 and 50 per cent saturation, thus 

 corroborating Avery's observations. 



