CALVIN B. COULTER 515 



extraction with isotonic saccharose solution an effective means for 

 separating sensitizer from cells. 



Michaelis and Davidsohn^^ have endeavored to relate the phenom- 

 enon of specific agglutination to the electrical properties of the react- 

 ing substances, but state that typhoid bacilli and immune serum 

 combine readily when the particles of both are negatively charged, 

 so that their union cannot depend upon an affinity due to opposite 

 electrical charge. They conclude further that specific typhoid agglu- 

 tination and precipitation are independent of the hydrogen ion 

 concentration — a conclusion which if applied to the general reaction 

 between antigen and antibody would seem at variance with the facts 

 related above. 



We have shown in an earlier paper^* that the agglutination of 

 sensitized sheep cells has an optimum at pH 5.3 at which point the 

 occurrence of agglutination is independent of the presence of elec- 

 trolyte. This point does not coincide with the isoelectric point of 

 the cells which was found for both the normal and sensitized cells 

 to be about pH 4.65, and the suggestion was made that the optimum 

 for agglutination is related to the isoelectric point of the immune 

 serum. The present work is an outcome of the earlier investigation 

 and is concerned with the relation of the hydrogen ion concentration 

 to the union of hemolytic sensitizer and cells. 



EXPERIMENTAL. 



The methods employed were similar to those already described. 

 To investigate the combination of sensitizer with cells in the absence, 

 as far as possible, of electrolyte, sheep cells were washed in four 

 changes of isotonic saccharose solution after washing in saline solu- 

 tion. The cells were made lip to 10 per cent by volume of the con- 

 centrated sediment in saccharose solution. A series of eight to ten 

 tubes was prepared, each containing 5 cc. of isotonic saccharose 

 solution (9.2 per cent) and varying amounts by drop addition of 

 n/10 NaOH or n/10 HCl. To each tube were then added precisely 

 0.1 cc. of undiluted immune rabbit serum and immediately afterward 



2^ Michaelis, L., and Davidsohn, H., Biochem. Z., 1912, xlvii, 59. 

 24 Coulter, C. B., /. Gen. Physiol., 1920-21, iii, 309. 



