H-2 ANTIGENS OF THE MOUSE 5I 



greatly enhanced activity and activity could not be detected in 

 the soluble fraction. By fractional precipitation of fluid with 

 water, or by fractional dialysis of fluid against buffers of low 

 ionic strength, it was found that the dialysis precipitate (DP) 

 fractions contained inactive material. The resolving power of the 

 method was poor, however, and whereas material giving haemag- 

 glutination inhibition at 50 (jLg./ml. could be obtained, there was 

 considerable loss of activity in adjacent fractions. The following 

 procedure was therefore adopted: fluid was diluted with 0-75 

 vol. of distilled water and left at 2-3° overnight. The insoluble 

 material which appeared was almost inactive and was removed 

 by being centrifuged, resuspended in water, dialysed and freeze 

 dried. The supernatant solution was dialysed against slowly 

 running distilled water for 40 hours at 2-3° and the insoluble 

 fraction, which carried most of the activity, was recovered by 

 being centrifuged in the cold. This fraction gave inhibition at 

 about 100 (JLg./ml. and was obtained in the following yields from 

 successive batches of BP8/C3H: 0-95 g., 2*0 g., i-68 g., 1*33 g. 

 The total weight of precipitable material obtainable from 

 original cell-free ascitic fluid is 3 • o g. (approximately 2 • 5-3 • 5 per 

 cent of the total dry weight), so that rather more than half of this 

 is usually obtained in the active DP fraction and the remainder 

 appears in the inactive 0*75 vol. water dilution precipitate. The 

 variation in the weights of products is due to diflerences in the 

 average weight of mice in the batches at the time of tumour 

 inoculation. Yields from CL2 and EL4 are, of course, smaller. 



When normal mouse serum is dialysed under similar conditions 

 the insoluble fractions amount to about 12 per cent of the total 

 material but no activity is present. 



The active DP fractions give suspensions in BS which are 

 moderately stable. Fractional centrifugation showed that all 

 activity was in a fraction which could be sedimented at 80,000^ 

 for two hours, leaving 80 per cent of the weight of material 

 in the supernatant fluid, and although activity at the level of 



