The Hemolytic Amboceptor 323 



turbed. Some prefer to give the corpuscles a third washing, 

 but it does not seem to be necessary. Of the remaining 

 corpuscular mass, 5 c.c. are added to 95 c.c. of salt solution 

 to make a 5 per cent, volume suspension, in which form they 

 are ready for use. As the corpuscles of healthy sheep thus 

 treated form a practically invariable unit, no titration or 

 other preliminary is needed before they are used. They 

 must, however, be used within twenty-four hours to secure 

 satisfactory results, as they tend to soften when kept and so 

 to lose their standard value. 



(5) The Hemolytic Amboceptor. As the validity of the 

 test depends upon the ability or inability of the complement 

 to dissolve the corpuscles, and as this can only be achieved 

 when appropriate amboceptors are added, the hemolytic 

 amboceptors must correspond to the kind of blood-corpuscles 

 employed in the experiment. As has been shown, the greater 

 number of investigators now employ sheep corpuscles, hence 

 must use such corpuscles as the antigen through whose stimu- 

 lation the amboceptors or antibodies are excited. 



The usual method of obtaining the amboceptor is in the 

 blood-serum of an experimentally manipulated rabbit. A 

 large healthy rabbit is employed for the purpose, and is 

 given a series of intraperitoneal injections of the 5 per cent, 

 suspension of washed and sedimented sheep corpuscles pre- 

 pared as above described. These injections are usually given 

 about five days apart, and the dosage is usually 5, 10, 15, 20, 

 and 25 c.c. respectively. 



A serum of higher amboceptor content may be prepared by 

 using a greater number of corpuscles, and for this purpose the 

 solid corpuscular mass thrown down by centrifugalization 

 after the second washing is employed. Of this, 2, 4, 8, and 

 12 c.c., diluted with just enough salt solution to make it 

 pass readily through the hypodermic needle, may be regarded 

 as appropriate doses, the intervals being the same, viz., five 

 days. The amboceptor content of the rabbit serum seems to 

 be greatest about the ninth or tenth day after the last injec- 

 tion. Much care must be taken to see that the injected fluid 

 is sterile and the operations performed under aseptic pre- 

 cautions, as the rabbits are easily infected and not infre- 

 quently die. They also seem prone to die after the last in- 

 jection, so that it is best to have more than one rabbit under 

 treatment at a time. 



When the appropriate time has arrived the rabbit is bled 



