The Hemolytic Amboceptor .293 



sedlmented sheep corpuscles prepared as above described. These 

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

 is usually 5. lOj iSi 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 whirKng 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- 

 sents itself is how successful titration of reagents that may all be 

 more or less variable can be effected. To achieve this it is necessary 

 to begin with those that can be assumed to be least variable and 

 work up to those that are most so. 



(i) The Sheep Corpuscles. — As these come from a healthy animal, 

 are always treated in precisely the same manner and used under 

 standard conditions of freshness, they can be looked upon as an in- 

 variable factor. One cubic centimeter of the 5 per cent, suspension 

 forms a good working quantity and constitutes the unit. 



(2) The Normal Guinea-pig Serum Containing the Complement. — 

 As this also comes from a normal animal, is always treated in pre- 

 cisely the same manner, and is also used under standard conditions 

 of freshness, etc., it may also be looked upon as a factor subject 

 to very slight variation. Of this serum, o.i cc. (i cc. of a i :io 

 dilution, made with physiological salt solution) forms the unit, or 

 workmg quantity. 



These two reagents, therefore, may be regarded as the standards 

 of measurement through which the titer of a third is made possible. 



(3) The hemolytic serum from the rabbit treated (vith the sheep 

 corpuscles. 



This is subject to very great variation, according to the treat- 



