H-2 ANTIGENS OF THE MOUSE 49 



cells had been removed showed diat specific activity was present 

 there also, apparently in soluble form. Although the level of 

 activity was low (inhibition of haemagglutination at 5-10 mg./ 

 ml.) this seemed a favourable starting material for isolation of 

 active material since ascitic fluid is similar to serum, and of simpler 

 composition than cell homogenates. 



Ascitic fluid recovered after growth of tumour for six to ten 

 days (BP8/C3H) had a lower level of activity than that recovered 

 after 13 days. The level seemed to decrease again from 14 to 16 

 days. Preliminary experiments indicated that fractionation of 

 ascitic fluid with ammonium sulphate gave little concentration of 

 activity in any fraction but that active material adsorbed non- 

 specifically on to precipitates formed at various levels of salt 

 concentration. Attempts to elute activity from diethylaminoethyl 

 (DEAE)-cellulose, carboxymethyl (CM)-cellulose, or starch (for 

 block electrophoresis) within reasonable limits of pH were 

 unsuccessful, and eluates of freeze-dried ascitic fluid contained 

 very little active material. 



The capacity for non-specific adsorption indicated the necessity 

 of finding some way of obtaining a substantial degree of purifica- 

 tion in the initial step if severe losses of active material were to be 

 avoided. A possibility of achieving this arose from the observation 

 that activity could be recovered in the fraction precipitated from 

 ascitic fluid by simple dialysis against distilled water. This also 

 showed that activity was associated with some particular mole- 

 cular fraction and led to the feeling that the behaviour in previous 

 experiments might be due to lipid being associated with the active 

 substance. 



Large-scale extraction 



Mice were used in batches of 1,000; LAN and BP8/C3H 

 peritoneal contents were recovered directly by suction into 

 aspirators, citrate being used as anticoagulant. During the 

 growth of EL4/C57 and CL2/BALB/C little free fluid was 



