338 COLLECTED STUDIES IN IMMUNITY. 



for the. union with the blood-cells. Such a possibility must always 

 be borne in mind. 



The technique of this separation at low temperatures is very 

 simple. The tubes containing the blood and the serum respectively 

 are cooled to by being placed in iced water or by packing in ice. 

 Thereupon the serum, in amounts which are not far either way from 

 the single solvent dose, is added to the blood. After being kept 

 at for two hours the mixture is rapidly centrifuged and the super- 

 natant fluid quickly removed. If desired, the sedimented blood- 

 cells can be washed with salt solution and then suitably suspended. 

 The decanted fluid is again mixed with blood-cells. For this pur- 

 pose, in order not to increase the total volume, one takes the blood- 

 cell sediment centrifuged from the required amount of the 5% sus- 

 pension. If a complete separation of amboceptor and complement 

 has been effected, it will be found that neither are the sedimented 

 blood-cells dissolved nor is the decanted fluid able to dissolve the 

 blood-cells added anew. It is then necessary to determine the pres- 

 ence of complement in the decanted fluid, which is done by adding 

 suitable amounts of serum inactivated by heating. Similarly the 

 amboceptor anchored by the blood-cells at low temperatures is demon- 

 strated by adding to the sediment the complement present in the 

 decanted fluid. 



The second and simpler method is that of inactivating the hsemo- 

 lytic serum by means of heat and then activating the amboceptor 

 by the addition of complement. In this the chief difficulty often 

 consists in the fact that a certain complement required in a par- 

 ticular instance is not contained in all sera, and further that the sera 

 which contain this particular complement often in themselves dis- 

 solve the blood-cells by means of a normal amboceptor. 



There are several ways of overcoming these difficulties. The 

 neatest method and one which is applicable in many cases consists 

 in selecting as the complementing agent the serum of that animal 

 species whose blood is being tested, as, for example, using guinea-pig 

 serum as complement for amboceptors acting on guinea-pig blood. 

 In such cases a solution of the blood-cells by means of the animal's 

 own serum is, of course, precluded. 



In all other cases one must make use of complementing sera 

 which are unrelated to the species of blood in question. One fre- 

 quently discovers sera for this purpose which do not in themselves 

 dissolve the blood-cells to be tested, as, for example, in reactivating 



