SEROLOGICAL METHODS 213 



Those interested in standardization of antitoxin by flocculation or by 

 in vivo methods should consult Wadsworth (1947). Quantitative aspects 

 of flocculation are discussed in Boyd (1947), Kabat and Mayer (1948), 

 and Cohn (1952). 



It should be pointed out that flocculation is characterized by solubility 

 of the antigen-antibody complex in both antigen and antibody excess and 

 is usually found with horse antibody like diphtheria antitoxin. The 

 precipitin reaction, on the other hand, displays solubility of the precipitate 

 only in an excess of antigen. This type of reaction is characteristic of 

 most rabbit antisera. The alpha procedure is usually employed for anti- 

 bodies of the latter type. 



The super nate test. In conjunction with the precipitin test, particu- 

 larly the quantitative method, it may be useful to test the supernate for 

 an excess of antibody or antigen. If the antigen is homogeneous, usually 

 one or more tubes will be found to contain neither excess antibody nor 

 excess antigen. This is called the "equivalence zone.'' It might be 

 expected that the region of optimal proportions would coincide with the 

 equivalence zone, and in many systems this is so; however, there are some 

 systems where the optimal ratio is outside the equivalence zone. 



Failure to find a definite equivalence zone indicates that two or mor'^. 

 overlapping precipitin systems are present, and this, in turn, may be used 

 as an indication that the antigen is not pure, a situation which is not 

 unlikely if crude bacterial extracts are being used as antigens. The 

 supernate test, then, may be used to indicate impurity of an antigen. It 

 does not necessarily follow from this, however, that if a good equivalence 

 zone is found, the antigen is pure. It may be, but additional evidence for 

 homogeneity should be furnished. 



To perform a supernate test, a precipitin test is set up in the ordinary 

 manner using the quantitative technique or the antigen dilution method. 

 After the tubes have been incubated a sufficient length of time, the pre- 

 cipitates are removed by centrifugation and the supernatant fluids are 

 poured into clean test tubes. Into a small test tube 0.2 ml of supernate 

 and 0.2 ml of fresh antiserum are placed. Into another tube 0.2 ml of 

 supernate and 0.2 ml of antigen are placed, using antigen that has been 

 diluted considerably to avoid inhibition due to antigen excess. A precipi- 

 tate in the first tube shows that an excess of antigen was present in the 

 supernate, a precipitate in the second tube shows that excess of antibody 

 was present, and no precipitation in either tube indicates either the pres- 

 ence of an equivalence zone or faulty technique. 



Precipitation in gels. The precipitation of antigen and antibody in a 

 gel has been used for the analysis of antigen mixtures by Oudin, Ouchter- 

 lony, and others (see Oudin, 1952). In the one-dimensional simple diffu- 

 sion procedure, one reagent, usually antibody, is mixed with agar and 



