154 ANTIGENS AND THE TECHNtC OF SERUM REACTIONS 
with its own antigen, whereas another antibody in the same immune 
serum may be present in excess of its homologous antigen, so it remains 
partly uncomlnned; also, the antigen similarly may be in excess of its 
specific antibody. Weil,^ using a nearly pure protein, crystallized egg 
albumen, found that the supernatant fluid obtained after removal of the 
precipitate formed by the mixing of egg albumen and its specific anti- 
body, contained either egg albumen or specific antibody, but never 
both. 
Opie^ showed the effect of dilution upon the precipitin reaction by a 
series of experiments, one of which is reproduced herewith. A rabbit 
was immunized to horse serum, and the blood of this rabbit was tested 
for precipitins. 
Horse serum. 
Antihorse serum 
from rabbit. 
1 to 10. 
1 to 100. 
1 to 1000. 
1 to 10,000. 
1 to 100,000. 
1 to 5 

+ + + 
+ + + + 
+ + 

1 to 10 

+ 
+ + + 
+ + 

1 to 15 

+ 
+ + 
+ + 

1 to 20 


+ + 
+ + 

1 to 25 


+ + 
+ + 

Ito50 


+ 
+ 

1 to 100 

_ 

+ 

1 to 125 
— 
_ 
_ 
+ 
_ 
1 to 150 
— 
— 
— 

— 
1 to 250 
— 
— 
— 

— 
1 to 300 
— 
— 
— 

From this experiment and others, Opie demonstrated that an excess 
of antigen dissolved the precipitate formed by the union of specific 
antigen and specific precipitating serum, and therefore the inhibition of 
specific precipitation (inhibition, or proprecipitin zone) in the presence 
of a high concentration of antigen, is due to the solvent action of the ex- 
cess of antigen. He showed furthermore that when a suitable constant 
amount of antigen is mixed with increasing quantities of immune 
serum, the maximum precipitate is found when the antigen is mixed 
with several hundred times its volume of immune serum, the number 
of volumes depending upon the strength of the serum. Also an excess 
of antigen is demonstrable in the supernatant fluid. He concluded that 
the presence of uncombined antigen and antibody in the supernatant 
fluid is best explained by the assumption of a multiplicity of antigens 
in such complex mixtures as blood serum or egg-white. 
These reactions, ncAerthelesS, have a marked specificity: The sera 
of animals immunized against casein of cows' milk, for example, will 
cause precipitation in clear solutions of this protein, but will fail to 
cause precipitation in solutions of casein from human milk. The sera 
of closely-related animals may contain small amounts of "group" 
' Jour. Immunol., 190G, 1, 19. ^ ibid., 1923, 8, 19. 
