28 G. LEJEUNE-LEDANT 



Serological method 



Antisera have been titrated principally by the haemagglutina- 

 tion technique (with shght modifications) in the presence of 

 dextran and absorbed human serum (Gorer and Mikulska, 1954). 

 Only these results are reported in this paper. 



The tubes used for this test are 7- 5 mm. in diameter and 65 • 7 

 mm. long. Reagents are mixed in the ratio of o- 05 ml. antiserum 

 in doubling dilution in i*8 per cent dextran (Glaxo Intradex 10 

 per cent, salt-free in 5 per cent glucose) to 0-05 ml. of a 2 per cent 

 suspension of CBA erythrocytes in 50 per cent v/v absorbed 

 human serum. The tubes are incubated for 90 minutes at 37° 

 and centrifuged at 600^ for 30 seconds. 



The degree of agglutination is observed macroscopically, 

 using gentle agitation, and is verified microscopically, particularly 

 to estabhsh end points. Two control tubes are regularly used: 

 in the first, antiserum is replaced by i • 8 per cent dextran ; in the 

 second, absorbed human serum is added. The specificity of the 

 reaction is tested with isologous red cells. 



Results 



Injection of living cells. As shown in Table I, high titres of 

 haemagglutinins are only produced by repeated injections of 

 living cells — either spleen cells or epidermal cells. In our experi- 

 mental conditions, a single injection of living spleen or epidermal 

 cells, although evoking transplantation immunity, is unable to 

 ehcit a detectable titre of haemagglutinins. Specificity of the 

 reaction is tested with isologous SA red cells, which give unvary- 

 ingly negative results. 



Injection of cell-free extracts. The cell-free extracts are injected 

 at a concentration which will induce a violent transplantation 

 immunity in a control animal, with an accelerated breakdown of 

 the skin graft. The amount of splenic extracts used corresponds 

 to 250 X 10^ spleen cells, and the amount of epidermal extracts, 

 which are much more active, to 20 x lo'^ epidermal cells. These 



