H-2 ANTIGENS OF THE MOUSE 47 



Serological methods 



In order to follow the progress of purification, the dextran- 

 normal human serum (NHS) method of inducing haemagglutina- 

 tion with H-2 antisera (Gorer and Mikulska, 1954) has been used 

 for inhibition tests. These have been described by Davies and 

 Hutchison (1961), but are briefly as follows. For whole cells, or 

 for insoluble but dispersible preparations, an equal volume of 

 suspension was added to all tubes of a series of antiserum dilutions 

 (prepared in i per cent dextran) and incubated. After centrifuging, 

 samples of the supernatant were reincubated with red cells (in 

 I : 2 NHS which had been inactivated and absorbed with similar 

 red cells) and haemagglutination read microscopically. The 

 difference between the haemagglutination titre thus obtained and 

 the titre of the unabsorbed antiserum was taken as the measure of 

 activity. 



For soluble preparations and those giving stable suspensions or 

 emulsions, antiserum was used in a small number of haemagglu- 

 tinating doses and the potential inhibitor diluted out to give an 

 inhibition titre. Dextran and NHS were also included in this 

 system. 



For following purification it is considered essential to use a 

 monospecific antiserum, because if different H-2 specificities are 

 carried by different chemical entities a separation of these on 

 fractionation would be obscured by a polyspecific antiserum. 

 Although it is possible that the H-2 "antigens" really represent 

 different structural features of one molecular complex, as for 

 example in the "O" somatic antigens of the Enterobacteria 

 (Davies, i960), in a system based on pseudo-alleles this is less 

 likely to be the case. Some of the antisera obtainable using the 

 four mouse strains available are shown in Table 1; relatively 

 monospecific sera for H-2-C, D, D^ D\ E, E^ F and K are thus 

 available without resorting to absorption methods. 



Agar diffusion was carried out as described by Crumpton and 

 Davies (1956). 



