THE ZONE PHENOMENON 



201 



The Zone Phenomenon. The Optimal Antigen- Antibody Ratios. 



We may specify the precipitin content of an antiserum by determining the 

 smallest amount that will give a visible precipitate with a standard amount of 

 antigen ; and the antigen content of a solution may be similarly specified . by the 

 smallest amount precipitating with a standard amount of antiserum. Both these 

 end-point methods, however, have a limited application, because there is no simple 

 relationship between the degree and speed of precipitation, and the relative con- 

 centration of the two reactants ; the end-points may vary widely with small 

 variations in the conditions of the titration. 



If we examine a series of tubes containing a unit volume of an antibody solution, 

 to which unit volumes of graded concentrations of antigen have been added, we 

 shall see an end-point with one of the diluter solutions of antigen. As the antigen 

 concentration increases, the precipitate appears sooner and in greater amount, 

 but beyond a certain point the speed of precipitation and the amount of precipitate 

 falls off with ijicreasing antigen concentration, and a point is finally reached at 

 which there is no precipitation at all. 



The characters of this zone of marked precipitation were first investigated in 

 detail by Dean and Webb (1926) using rabbit antisera prepared against horse 

 serum protein antigens. Table 22 illustrates their results. It shows the degrees 

 of reaction 32 minutes after mixing increasing dilutions of antigen with a constant 

 1 : 20 dilution of a particular rabbit antiserum. 



TABLE 22 



Showing the State of reacting Mixtures 32 Minutes after mixing a 1 : 20 Dilution of 

 Antiserum with increasing Dilutions of Antigen (Horse Serum) 



Flocculation is most advanced in the tube containing equal volumes of 1 : 20 

 antiserum and 1 : 320 of antigen. In terms of the original rabbit antiserum and 

 the solution of horse serum proteins, the ratio of antigen to antibody in this tube 

 is 1 : 16. Mixtures in which this ratio is departed from, in the direction either 

 of excess or deficiency of antigen, flocculate more slowly or not at all. 



Dean and Webb called the antigen-antibody ratio that gave most rapid floccula- 

 tion, the optimal 'proportion or the optimal ratio. For a given antiserum and 

 antigen, they found that the ratio was constant for all dilutions of antiserum. 

 In the example quoted, repetition of the titration with a 1 : 40 dilution of antiserum 

 gave the most rapid flocculation with a 1 : 640 dilution of antigen, and a 1 : 10 

 dilution was optimal with a 1 : 160 dilution of antigen. 



