208 METHODS OF SERO-DIAGNOSIS. 



The " complement fixation " method of diagnosis is based 

 on the idea that if an antigen is brought into relation with its 

 suitable specific antibody when the latter is present in an in- 

 activated serum, and complement be added, then this complement 

 becomes fixed to the antigen through the medium of the specific 

 antibody and hence, being fixed, is no longer available to reactivate 

 a previously inactivated hemolytic serum, which is added in the 

 presence of a suspension of the corpuscles against which this 

 hemolytic serum is prepared. 



Thus it will be seen that for the performance of test there 

 is necessary : — 



(a) The antigen. This may be a bacterial extract, or a 

 solution containing the products formed by the organ- 

 ism in its growth, or, perhaps, an extract of an organ 

 in which the causal agent is present. 



(b) The serum of the subject to be examined. 



(c) The complement, obtained by using the recently- 

 obtained serum of some suitable animal. 



(d) The hemolytic serum obtained by injecting repeatedly 

 an animal of one species with the blood of that of 

 another species. Sometimes the haemolysins normally 

 present in the serum of the subject to be examined are 

 made use of instead. 



(e) A suspension in a suitable fluid, such as physiological 

 salt solution (.85 % to .9 % sodium chloride in water) 

 of the red corpuscles, against which the action of the 

 hgemolytic serum is directed. 



In describing the method of application of the " complement 

 fixation " test, it is perhaps best to take a special example as an 

 illustration, say, that of the diagnosis of glanders. 



In this case the hemolytic serum used is usually that of a 

 rabbit which has been repeatedly injected with the blood of 

 another animal, which is very often a sheep. Blood is taken 

 from the jugular vein of the sheep, defibrinated, and must he 

 washed repeatedly in a physiological salt solution so as to remove 

 any serum adherent to the corpuscles, as otherwise antibodies 

 reacting with this serum might be produced which would interfere 

 with the test. When, after repeated injections, it is thought that 

 the rabbit serum is hgemolytic to a high degree, it is obtained 

 either by bleeding the animal from the ear or by bleeding it to 

 death from the carotid artery, and then it is tested in order to 

 find the smallest amount which is capable of producing the 

 haemolysis of a given amount of corpuscles. 



In the titration of the serum the following method is used : 

 If the rabbit serum is fresh at the time of testing, it must be in- 

 activated by heating it in a water bath for half an hour to one 

 hour at 56 C before the test is applied. 



A series of test tubes is then taken and a fixed quantity, 

 usually 1 cc of the inactivated rabbit serum in different dilutions, 

 say, 1-10 to 1-5,000, is placed in each tube. Then there is added 

 to each tube an excess of complement, most often a quantity of 

 0.5 cc of a 1-10 solui^on of guinea-pig serum in physiological salt 



