SEROLOGICAL METHODS 217 



guinea-pig serum to each tube. Then place 1.7 ml of saline solution and 

 0.5 ml of 2 per cent erythrocyte suspension into each tube. The contents 

 of the tubes are mixed by shaking, and the rack incubated in a 37°C water 

 bath for 1 hr. The unit of hemolysin is taken as the highest dilution that 

 yields complete hemolysis. A satisfactory hemol3^sin should yield a fairly 

 high titer. The unit should be 1 : 4,000 or higher. 



To prepare sensitized erythrocytes, place the desired volume of 2 per 

 cent erythrocytes in a flask and add an equal volume of hemolysin. 

 Incubate at room temperature for 30 min. The dilution of hemolysin 

 used should contain two units as calculated above; therefore, if the unit 

 were found to be 1 : 4,000, one would select the 1 : 2,000 dilution for sensi- 

 tization. Sensitized erythrocytes should not be kept from one day to the 

 next. 



Complement. The serum from nonimmunized, healthy guinea pigs is 

 used as a source of complement. Since the serum from some animals may 

 be low in complement activity, it is necessary to pool the serum from at 

 least five animals. Commercially prepared lyophilized complement may 

 be used; however, occasional lots are unsatisfactory. 



The guinea pigs are bled from the heart or anesthetized and exsangui- 

 nated by cutting the large blood vessels on both sides of the neck. The 

 animals should not be fed for at least 12 hr prior to bleeding. 



The blood is allowed to clot in centrifuge tubes, and the serum is centri- 

 fuged until free from cells. Serum which appears slightly turbid may 

 contain a few cells and should be recentrifuged. Since complement is 

 destroyed rapidly by higher temperatures, ^ it is best stored in the frozen 

 or dried (lyophilized) state. A convenient method is to place 1.0- to 

 5.0-ml aliquots of the pooled serum in tightly stoppered vials. These are 

 then stored in the deep freeze until needed. For the complement titra- 

 tion, a vial is removed from the deep freeze, thawed in cold water, and 

 diluted to 1 : 30 with, cold saline. The diluted serum should be kept in the 

 cold until needed and should be discarded if not used within a few hours. 



In dealing with systems where the correct antigen ''dose" has been 

 established, it is customary to add the optimal amount of antigen (see 

 next section) to each tube in the complement titration. This will offset 

 any slight anticomplementary effect of the antigen. 



Using a 0.2-ml pipet graduated in 0.001 ml, place the following amounts 

 of 1 : 30 guinea-pig serum in a series of 15- by 85-mm test tubes : 0.20, 0.25, 

 0.30, 0.35, 0.40, 0.45, 0.50 ml. Add sufficient saline to each tube to bring 

 the volume to 1.5 ml (1.3 ml in the first tube), and then place 0.5 ml of the 

 proper antigen dilution into each tube, shake the rack, and incubate in a 

 37°C water bath for 1 hr. This incubation period will allow time for the 



^ Complement is also inactivated by passage through Berkefeld and other types of 

 sterilizing filters. 



