122 THE PRINCIPLES OF IMMUNOLOGY 



pletion of hemolysis it can be demonstrated that the absolute amount 

 rather than the degree of concentration is of importance in regard to 

 the amboceptor. 



This may be shown by placing in each of four tubes 0.5 c.c. 5 per cent, 

 suspension of corpuscles and adding to the second, third and fourth tubes, 

 respectively, four, nine and fourteen volumes of salt solution. To each of the 

 four tubes is added one unit amboceptor, and the mixture incubated at 37 C 

 for one-half hour to permit absorption of amboceptor. The tubes are cen- 

 trifuged and the supernatant fluid is discarded. To each tube is added i.o c.c. 

 complement so diluted as to contain one unit, and the mixtures again incu- 

 bated at 37 C. for one hour. Hemolysis will occur equally in all tubes 

 showing that the complete absorption of amboceptor by the cells occurred 

 in spite of marked dilution in some of the tubes. 



Rate of Absorption of Amboceptor. The absorption of ambo- 

 ceptor varies in rapidity under different conditions. For example, 

 absorption takes place more readily at 37 C. than at 20 C., and more 

 readily at 20 C. than at o. An exception to this rule appears in 

 cases of paroxysmal hemoglobinuria. Some of these cases possess in 

 the blood an autohemolysin which does not enter into combination with 

 erythrocytes at body temperature. If the blood is withdrawn and 

 placed at a temperature of o to 10 C. for an hour the cells absorb the 

 amboceptor and subsequent incubation at 37 C. permits the inter- 

 action of complement so that hemolysis results. With this and 

 possibly some other exceptions the general rule holds true that tem- 

 peratures approaching 37 C. favor the union of amboceptor and antigen. 

 Certain physical conditions also play a part in rapidity of absorption 

 as may be shown by the following experiment in which the mixture of 

 corpuscles and amboceptor is made under different conditions. 



Two wide test tubes or small beakers are marked A and B. In A are 

 placed six units of cell suspension; namely, 3.0 c.c. 5 per cent, suspension. 

 To this are added drop by drop 3.0 c.c. amboceptor, so diluted that it con- 

 tains six units, the tube being shaken constantly during the addition. In 

 tube B the process is reversed, the amboceptor being placed in the tube and 

 the cell suspension added drop by drop. These mixtures may be titrated 

 against varying amounts of complement in a series of tubes, or six units of 

 complement may be added to tube A and tube B. An hour's incubation at 

 37 C. will show less active hemolysis in tube B than in A. The probable 

 explanation is that the first cells added to the amboceptor in tube B absorb 

 all or nearly all the amboceptor, and the subsequently added cells are only 

 partly saturated or take up no amboceptor at all. 



This experiment illustrates the very rapid absorption of amboceptor 

 by cells and also the fact that cells may absorb considerably more than 

 one unit of amboceptor. 



Dissociation of Amboceptor-Antigen Union. Whereas tempera- 

 tures up to 37 C. appear to favor absorption of amboceptors, Bail, 

 Tsuda and others have shown that a temperature of 42 C. results in 

 a partial dissociation of amboceptor. That dissociation of amboceptor 

 and cells could occur was shown independently by Muir and by Mor- 

 genroth. Muir mixed i.o c.c. 5 per cent, corpuscle suspension with 

 ten units amboceptor and allowed the mixture to stand at room tem- 

 perature for one hour. The tube was then centrif uged, the corpuscles 



