The Precise Measurement of Hemolysin

The content in hemolysin of a solution is commonly measured as the amount which is just sufficient to produce complete hemolysis of an arbitrary quantity of red blood cells, usually 0.5 cc. of a 5 per cent suspension of washed erythrocytes. The method employed for such measurement gives results which are neither exactly comparable in determinations made at different times, nor highly precise. The susceptibility of erythrocytes to hemolysis is influenced by many factors, and the amount of the minimal hemolyzing quantity must vary accordingly for every specimen of test cells. Lack of precision in the measurement results from the fact that relative differences in hemolysin content between adjacent tubes in a titration series must be great, in order to distinguish the end-point, so that the value determined differs often by a large amount from a possible true value. The method of titration of hemolysin described in this paper was developed for a study of the association of hemolysin with different fractions of immune serum and plasma protein. It measures hemolysin content as the ratio of the hemolytic activity of a given solution to that of standard immune serum. This standard has been usually a portion of the whole serum from which isolated protein fractions have been derived. This choice of a standard immune serum instead of a given quantity of erythrocytes, as the unit of measurement of hemolysin content, has the advantage that applies to the use of a diphtheria antitoxin for the standardization of toxin and of new antitoxin: the antibody is the most stable biological element of the immune system. The necessity for a large increment of hemolytic substance in successive tubes in a titration to determine the minimal hemolyzing quantity has long been recognized, and is not peculiar to the immune