SEROLOGICAL METHODS viii,v-19 



Tubes 4, 5 and 6 should show complete hemolysis, indicating that 

 the serum and antigen are not anticomplementary and that the 

 iieraolytic system is working properly. 



There should be no hemolysis in tube 7, showing that the salt 

 solution is isotonic with the sheep cells. When several tests are made 

 at the same time with the same immune serum, the control tubes 

 4, 6 and 7 need not be repeated. It is necessary, however, to add an 

 anticomplementary antigen control whenever a different antigen is 

 used, and another anticomplementary serum control whenever a 

 different serum is used. 



Example: A specimen protocol, giving amounts of the reagents 

 presumed to have been decided upon after the preliminary titrations 

 described above, is given in Table 6. (N. B. The amounts stated here 

 are arhitrary amounts and are not to he applied to an actual test unless 

 justified by previous titrations.) 



A measurement of the titer of an immune serum can be made by 

 this test. The specificity of the serum can be judged only by testing 

 it in this manner against other antigens. In interpreting the results 

 of this test for the purpose of pure culture studies, it may be assumed 

 that when an organism causes complement fixation in any of the 

 tubes 1 to 3 with an antiserum produced by the immunization of an 

 animal against another organism, the two organisms have common 

 antigens. The results of complement fixation tests, however, must 

 not be regarded as a basis for exact determinations of identity, as 

 certain antigens may show positive reactions with the sera of entire 

 groups. The test is often more indicative of group relationships 

 than of identities. 



TITRATION OF TOXINS, TOXOIDS AND ANTITOXINS 



(Flocculation method) 



An unknown toxin or toxoid may be titrated with an antitoxin of 

 known value or an unknown antitoxin with a toxin or toxoid of known 

 value in vitro. In serial mixtures of the two, there first occurs a 

 cloudiness followed by a precipitate in some of the tubes, and finally 

 a definite flocculation in one tube which is taken as the tube contain- 

 ing the "indicating mixture". The flocculation in this flrst tube 

 may be followed by flocculation in other tubes about it within a short 

 time. The ''indicating mixture" however, is alioays the initial tube 

 to flocculate and must be watched for rather cautiously. From this 

 "indicating mixture" is calculated the floccidating unit of the toxin, 

 which has been designated Lf. The Lf may be defined as the amount 

 of toxin equivalent to 1 unit of antitoxin as established by flocculation. 

 There is no complete agreement or relationship between the M.L.D., 

 Lo, L, and the Lf values of a toxin. The first three of these units 

 have been designated as "in vivo units" and the fourth as an "m 

 vitro unit". Flocculation may occur at any temperature up to 55°C., 

 above which the reaction becomes irregular and often completely in- 

 hibited. A temperature of 40° to 50°C. is the most suitable zone. 

 The time of incubation and of flocculation vary with different toxins 



