MODIFICATIONS OF THE WASSERMANN REACTION 451 



2. Human Corpuscles. These are washed three times with normal 

 salt solution, and used in dose of 1 c.c. of a 1 per cent, suspension. 

 To a graduated centrifuge tube containing 9 c.c. of sterile 2 per cent, 

 sodium citrate in normal salt solution add 1 c.c. of blood and shake 

 gently. This amount of blood is easily secured by pricking the finger 

 with a lancet and collecting the blood in the centrifuge tube up to the 

 mark 10. This is centrifuged thoroughly, and the supernatant fluid 

 drawn off. More saline solution is then added to the corpuscles stirred 

 up and the mixture centrifuged. The washing is repeated once more, 

 and the supernatant fluid discarded. The corpuscles are then sus- 

 pended in sufficient salt solution to make a total volume of 100 c.c. 



3. Hemolytic Amboceptor. Antihuman hemolysin is prepared by 

 immunizing rabbits with human cells, as described on p. 72. It is a 

 difficult matter to secure a potent amboceptor; human erythrocytes 

 are more toxic than sheep's cells for rabbits, and most animals produce 

 but small amounts of the amboceptor. Hemagglutinins are produced in 

 large amounts, and when using a low titer hemolytic serum, the test- 

 corpuscles are quickly agglutinated in small dense masses that are 

 broken up with difficulty and that interfere greatly with hemolysis. 

 With serums having a titer of 1 : 1000 or over, the agglutinins are not 

 so much in evidence; a satisfactory reaction is best observed, therefore, 

 with a potent amboceptor (1 : 1000) serum. 



The hemolytic serum may be preserved in 1 c.c. ampules after 

 adding an equal part of glycerin, and a stock dilution prepared and 

 titrated in the usual manner. The serum is also well preserved dried 

 on filter-paper, as described on p. 79. A trial titration should always 

 be made to determine the potency of the serum before the paper slips 

 are prepared. 



// paper amboceptor is used, the uniform rule of titrating it with the 

 complement and corpuscles on hand should be observed before the actual 

 tests are made. This is done chiefly because, where one guinea-pig 

 serum is used for complement, it may occasionally happen that the 

 serum is less active than usual, so that if fixed doses of complement and 

 amboceptor are used, the reactions may at times prove to be incomplete 

 and inaccurate. The process of titration is so simple that any one may 

 readily conduct it, and thus fulfil the most important requirement of any 

 complement-fixation test, namely, adjustment of the complement, 

 amboceptor, and corpuscles to one another. 



Titration of Serum Hemolysin. Prepare a 1 : 100 dilution by mixing 

 0.1 c.c. of immune serum (inactivated) with 9.9 c.c. of saline solution. 



