METHODS OF STUDYING H^EMOLYSINS. 337 



(6) Thermic and chemic influences, such as heating to 50-60 C., 

 the action of alkalies and acids, digestion with papayotin. 



The separation of amboceptor and complement at low tempera- 

 tures is of the utmost importance and has been used for the analysis 

 of complex hsemolysins with considerable success. The conditions 

 necessary for the successful operation of this method have been 

 discussed in detail in a previous paper. A separation is only then 

 possible when at low temperatures the affinity between the cyto- 

 phile group of the amboceptor and the receptor is greater than that 

 between the complementophile group of the amboceptor and the 

 corresponding group of the complement. The degree of difference 

 in the affinities would, of course, determine the degree of complete- 

 ness of the separation. In some instances most peculiar relations 

 are found, as is shown, for example, by the behavior of eel serum 

 to rabbit blood. Attempts to effect separation at low temperatures 

 fail in this case, first, because haemolysis ensues even at C., and 

 second, because the employment of higher concentrations of salt 

 (up to 5%), which in other cases has afforded a means of loosening 

 the combination of amboceptor and complement, does not suffice 

 to prevent haemolysis. Naturally from this behavior w r e must not 

 conclude that eel serum does not contain a complex heemolysin, but 

 merely that in this case peculiar conditions are present which, owing 

 to the insufficiency of the methods thus far employed, are still obscure 

 to us. In those cases in which separation at low temperatures fails, 

 a, second method may be considered. This depends on the fact 

 that a high degree of salt concentration, somewhat after the manner 

 of low temperatures, can prevent haemolysis; concentrations which 

 still permit the union of receptor and amboceptor preventing that 

 of amboceptor and complement. The prevention of ha?molysis by 

 means of salts, first described by Markl l and erroneously ascribed 

 by him to conditions of diffusion, is also due to this. Markl entirely 

 overlooked the fact that in certain cases the combination of toxin 

 and antitoxin (e.g. Tetanus toxin + Antitoxin) is also .prevented by 

 salt. (Knorr.) For the application of this method see Ehrlich and 

 Sachs, page 214. 



It is perfectly obvious that the cold method will fail absolutely 

 in cases like the one described by Ehrlich and Sachs (page 217) in 

 which the union of amboceptor and complement is the prerequisite 



1 Markl, Zeitschr fiir Hygiene, Vol. 39, 1902. 



