H-2 ANTIGENS OF THE MOUSE 53 



on to gradients running from lo per cent to 40 per cent tartrate 

 (density i-o6 to 1*23). After centrifuging at 95,000^ to equili- 

 brium, a number of bands appeared which were recovered 

 separately, and dialysed free from salt. Samples of solution were 

 tested for activity and the remainder freeze dried for analysis. 

 All but two bands were inactive and subsequently the fractions 

 were recovered as follows: top fraction (TT), upper middle band 

 (TMi), lower middle band (TM2) and bottom fraction (TB). The 

 middle fractions together account for 60-80 per cent of the SP 

 (Spinco precipitate) fraction. Typical analyses are shown in 

 Table III, where most figures are averages from at least ten 

 experiments. 



Both the TMi and TM2 fractions had the property of inhibiting 

 in the haemagglutination test, but only TM2 fractions induced 

 haemagglutinating antisera when injected in appropriate allo- 

 geneic mouse strains. 



When TM2 fractions are recycled through new tartrate 

 gradients material is again obtained in the TT and TB regions. 

 This suggests that the active material is not stable in strong salt, 

 and although the data given below are for tartrate gradient 

 materials, sucrose gradients are now being used over the same 

 density range for increased stability. For these gradients activity 

 is found in a central band (SM) and the material recovered, after 

 dialysis to remove sucrose, gives the same analytical figures as 

 TM2 fractions (Davies, 1962). 



Hazards of contamination 



It is not practicable to recover the products from batches of 

 1,000 mice under sterile conditions and from time to time 

 organisms have appeared which grow readily at low temperature 

 and are resistant to antibiotics. Various means of sterihzation 

 tested have proved harmful to the H-2 antigen, except u.v. 

 irradiation. After the first sedimentation in the Spinco, the 

 resuspended precipitate is therefore distributed in dishes to a 



