50 OHNO. 



In the antephase of agglutination, the serum, even though it contain 

 a great quantity of agglutinin, can agglutinate only that quantity of 

 agglutinable substance which is not picked up by agglutinoicl, as the 

 latter unites more readily than agglutinin with agglutinable substance; 

 therefore, the proportions of agglutinin uniting with agglutinable sub- 

 stance during the antephase do not show the usual regular proportions. 

 However, . the regular ratio between agglutinoid and agglutinable 

 substance can be shown by subtracting the available agglutinable sub- 

 stance, as indicated by the degree of agglutination, from the amount 

 of agglutinable substance introduced into the test tube. The variations 

 in the quantity of agglutinable substance united with agglutinin at 

 different serum dilutions is, therefore, not a proof of different grades of 

 absorption as Bisenberg and Volk consider it to be, but is dependent 

 upon the presence of agglutinoid. 



The rate of conversion of agglutinin to agglutinoid varies with the 

 temperature and the dilution at which the agglutination test is performed, 

 being greater at a high temperature and in high dilution. Eisenberg 

 and Volk did not pay attention to this fact, although it is of im- 

 portance in considering a theory of agglutination. 



I accept the view first advanced by Bordet that agglutination may be 

 divided into a stage of fixation of agglutinin by bacilli, and a stage of 

 aggregation of bacilli united with agglutinin. However, according to 

 the results of my experiments, three and one-half to four and one-half 

 hours at 37° C. or four and one-half to five and one-half hours at room 

 temperature (first two hours at 37° C.) is necessary for the fixation of 

 agglutinin to bacilli. The time required for aggregation is much longer 

 than that needed for fixation, and if we do not allow sufficient time for 

 aggregation to affect all the quantity of fixed agglutinable substance, we 

 will not be able to form an accurate idea of the degree of agglutination 

 produced by any given amount of serum. My experiments indicate that 

 it requires the time limits given above for a complete union and from 

 twenty to twenty-four hours at 37° C. for complete aggregation. As 

 Eisenberg and Volk conducted their tests for only two hours at 37° C. 

 followed by twenty-two hours at room temperature, I estimate that they 

 recognized only three-fifths of the real value of their serum; therefore, 

 the quantity of absorbed agglutinin, calculated by Eisenberg and Volk, 

 is larger than the real quantity by a certain amount of agglutinin and 

 agglutinoid, which is to be given by the expression : 

 (f r'+d') AgE— AgD 



Where r'=the quantity of agglutinin remaining ununited; 



d'=number of units of agglutinin degenerating during the test; and AgE 

 and AgD represent respectively the agglutinin and agglutinoid units 

 of Eisenberg and Volk. 



