1 62      Chemical  Reactions  of  Diphtheria  Antitoxin.  { Am-  Jgjr- 
to  250  Cc.  One  to  three  Cc.  were  tested.  One  Cc.  protected  against 
one  L  -f-  dose.  Presumably  the  neutralizing  power  was  even  greater 
than  shown.  The  colloidal  solution  obtained  with  0.85  per  cent. 
NaCl  was  diluted  to  350  Cc.  Even  one  Cc.  protected  against  one 
L  +  dose  of  the  toxin.    Some  antitoxin  went  into  both  preparations. 
Three  Cc.  of  globulin  preparation  III  were  precipitated  over  night 
with  a  saturated  aqueous  solution  of  picrolonic  acid.  After  centrif- 
ugalizing  the  clear  solution  was  shaken  several  times  with  iso-butyl 
alcohol.  The  alcohol  gave  no  precipitate.  The  colorless  solution 
was  made  up  to  250  Cc.  Even  3  Cc.  of  this  did  not  protect  against 
one  L  +  dose  of  the  toxin.  Evidently  the  filtrate,  i.  e.,  the  centrif- 
ugalized  solution  after  picrolonic  acid  contained  no  antitoxic  units. 
This  highly  concentrated  globulin  corresponded  in  its  reaction  to 
picrolonic  acid  to  preparation  I  which  was  of  a  lesser  concentration. 
The  precipitate  from  picrolonic  acid  was  shaken  with  NaCl  (0.85 
per  cent.)  and  made  into  a  suspension.  This  was  shaken  several 
times  with  iso-butyl  alcohol  to  remove  picrolonic  acid.  The  isobutyl 
alcohol  precipitated  a  gelatinous  mass,  which  after  centrifugalizing 
became  colorless  on  further  shaking  with  iso-butyl  alcohol.  This 
white  gelatinous  material  was  dissolved  in  NaCl  (0.85  per  cent.)  by 
the  addition  of  NaOH  n/10  and  the  corresponding  amount  of  n/io 
HC1  was  then  added.  This  colloidal  solution  was  made  up  to  500 
Cc.  with  NaCl  (0.85  per  cent.).  This  solution  had  antitoxic  value, 
but  did  not  correspond  to  the  full  number  of  units  used.  This  may 
perhaps  have  been  due  to  the  long  contact  with  iso-butyl  alcohol. 
The  centrifugalized  solution  after  iso-butyl  alcohol  precipitation 
was  made  up  to  100  Cc.  but  even  3  Cc.  did  not  protect  against  one 
L  -\-  dose,  so  that  it  contained  few  if  any  antitoxic  units.  Evidently 
iso-butyl  alcohol  precipitates  antitoxin  at  least  from  this  prepara- 
tion. 
Two  Cc.  of  preparation  III  (8,000  antitoxic  units)  were  precipi- 
tated with  a  saturated  aqueous  solution  of  picrolonic  acid  and  the 
precipitate  was  shaken  several  times  with  NaCl  (0.85  per  cent.)  cen- 
trifugalized, then  the  precipitate  shaken  again,  then  filtered  through 
filter  paper.  The  filtrate  was  then  made  up  to  750  Cc.  Even  3  Cc. 
did  not  protect  from  one  L  -f-  dose  of  the  toxin.  The  loss  of  activity 
may  have  been  due  to  the  filtering  through  filter  paper,  or  to  the 
preparation  having  stood  several  days,  but  it  was  thought,  as  there 
was  picrolonic  acid  present,  that  this  should  preserve  it. 
10  Cc.  globulin  preparation  I,  diluted  with  an  equal  volume  of 
