172 
Dourine 
the sera are tested without antigen, as in the serum controls, the dourine 
sera, of course, give no specific reactions, but the inhibition reactions 
are given by normal and dourine sera alike when insufficiently inacti¬ 
vated, though to a lesser degree than when antigen is present. 
Conclusion. Suspected horse serum must be heated to at least 
58° C. (59-60° for safety) and mule or donkey serum to 62° C., to 
eliminate non-specific reactions. 
The Antibody content of Dourine Sera, and the dose of suspected 
Serum necessary for a Diagnostic Test. 
The maximum dose of horse serum used in a diagnostic fixation 
test is 0-2 c.c. This amount is not exceeded for fear of any disturbance 
to the haemolytic system by the non-specific reactions which larger 
doses are apt to cause. Double the amount can actually be used with 
perfect safety provided the serum is correctly inactivated. But it is 
unnecessary to use more than 0-2 c.c., for that amount of serum of a 
dourine horse will contain in the case of a serum very weak in antibody 
content at least one unit, and in the case of a serum strong in antibody 
ten, twenty, forty or more units—and one unit of antibody is sufficient 
to give a positive reaction with the fixation test. 
That this is so may be determined by taking a series of sera collected 
from animals in active and in latent phases of the disease and titrating 
out each serum for antibody content. 
The experiment is carried out as follows: 
The sera are first inactivated by heating for one half hour at 59° C. 
Three stock tubes are then taken for each serum, (1) containing the 
pure serum, (2) a dilution of 1: 10, and (3) a dilution of 1: 100, these 
dilutions permitting of the accurate measurement of the smaller doses. 
Twelve tubes are now arranged for each serum to be tested—the 
first and last to contain 0-2 c.c. of undiluted serum, the largest amount 
used in the test, the last tube being the serum control without antigen, 
the intervening tubes to contain gradually decreasing doses of serum. 
Enough salt solution is then added to make up to TO c.c. in each tube, 
then the antigen and complement in amounts previously determined 
by careful titration, and finally, after incubation for seventy minutes, 
haemolytic serum and red cells—as in a diagnostic test. 
An experiment of this kind is given below, the titres of seven sera 
from different horses in different phases of the disease being determined. 
