SEROLOGICAL METHODS 



viiij7-15 



The serum is allowed to separate from the clot, pipetted off, and treated with 0.4% 

 phenol, 0.3% tricresol, or an equal amount of 50% neutral glycerol. The potency will 

 be retained for many months, when stored in the refrigerator. Titrations should be 

 made at intervals, however, not exceeding three or four months. 



The titration of hemolytic amboceptor, using a constant amount of complement, is 

 discussed below. The hemolytic titer (unit) should be at least 0.25 ml. of a 1-1000 

 dilution. 



If the amboceptor does not have such potency as this, it is advisable to continue 

 the injection of increasing amounts of the sheep cells. For sharp reactions, in which 

 a minimal amount of complement can be used, and to have an amboceptor which can 

 be diluted well beyond its agglutinative effect upon red corpuscles, it is advisable to 

 prepare an amboceptor with a high titer. 



TITRATION OF REAGENTS 



Before proceeding with the test, the relative strength of each of 

 the reagents must be known and the amounts necessary for a suc- 

 cessful test determined. This process is known as titration. A.11 the 

 reagents, with the exception of the red corpuscles, and the specific 

 immune serum (antibody), should be titrated before any test is 

 conducted. Whenever a freshly prepared reagent is used, it must 

 be titrated. Daily titrations of complement must be made when 

 tests are done each day. 



Titration of Amboceptor {Hemolysin). In this titration, decreasing amounts of am- 

 boceptor are mixed with a constant amount of complement and added to sheep's red 

 corpuscles to determine the smallest amount of amboceptor which will cause hemolysis 

 of the sheep cells. (To prepare a specimen of complement having good average proper- 

 ties, mix the blood serum obtained from bleeding at least 3 normal guinea pigs.) Dilute 

 this complement 1 to 10 with saline. It is advisable to keep the flask containing 

 complement on ice or in ice water, to prevent the deterioration which takes place 

 appreciably, even at room temperature. Next make up the following series of dilutions 

 of the anti-sheep amboceptor: 1-100, 1-200, 1-400, 1-1600, 1-3200, 1-6400. Prepare 

 a 2.5% suspension of washed red corpuscles (sheep) as described above. Set up the 

 tubes for this titration according to the following protocol. (Table 3) 



TABLE 3 



TITRATION OF HEMOLYTIC AMBOCEPTOR WITH RESULTS IN A TYPICAL INSTANCE 



Tubes 7 and 8 are controls used to show whether or not either the amboceptor or com- 

 plement is hemolytic. If either is hemolytic, that reagent should be discarded. Some 

 specimens of complement are quite hemolytic. 



After the mixtures are made, place the rack containing the tubes in the water bath 

 at 37° C. and incubate them for 15 min., shaking repeatedly. At the end of the period 

 of incubation, note hemolysis. The tube containing the highest dilution of the ambocep- 

 tor which produces complete hemolysis of the cells (tube 5 in instance illustrated in 

 Table 3) denotes the titer of the amboceptor. In this system, 0.25 ml. of that dilution 

 of the ambocej)tor is called one iniit of the amboceptor. This unit noiv becomes a fixed 

 standard, as the amboceptor is a stable substance. In subsequent titrations of comple- 



