284 Wassermann Reaction for Diagnosis of Syphilis 



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

 mass, 5 cc. are added to 95 cc. 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 prehminary is needed before they are used. 

 They must, however, be used within seventy-two hours to secure 

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

 their standard value. If kept longer than twenty-four hours they 

 should be washed before u^ing. 



(s) The Hemolytic Amboceptor. — As the validity of the test de- 

 pends 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 stimulation 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 intra- 

 peritoneal injections of the 5 per cent, suspension of washed and 

 sedimented sheep corpuscles prepared as above described. These 

 injections are usually given about five days apart, and the dosage 

 is usually 5, 10, 15, 20 and 25 cc. respectively. 



A serum of higher amboceptor content may be prepared by using 

 a greater number of corpuscles, and for this purpose the solid cor- 

 puscular mass thrown down by centrifugalization after the second 

 washing is employed. Of this, 2, 4, 8, and 12 cc, 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 injection. Much care^ must be taken to see that the injected 

 fluid is sterile and the operations performed under aseptic precau- 

 tions, as the rabbits are easily infected and not infrequently die. 

 They also seem prone to die after the last injection, 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 from 

 the carotid artery, according to the directions given in the chapter 

 upon Experiments upon Animals. 



The blood thus obtained is permitted to coagulate, and the serum, 

 which should be clear, removed with a pipette. More serum may be 

 obtained from the clot by cutting it into strips, placing these in a 

 centrifuge tube, and whirling them for fifteen minutes. 



Having thus described the preparation of the reagents to be em- 

 ployed in making the Wassermann test, the next step, that of titrat- 

 ing them, becomes essential. One of the first questions that pre- 



