SEROLOGICAL METHODS viii^,-!! 



and sterilization of the antigen may be done by filtration (Berkefeld). 

 High titered sera have been produced by injecting progressively in- 

 creasing doses of antigen at 3-day intervals. After 5-6 injections, 

 a test bleeding is made and if the titer is low additional injections are 

 given. Bleedings are made a week after the last injection. When a 

 sufficiently high titer has been reached, the rabbit is bled aseptically 

 from the heart ;^ the blood is allowed to clot; and the clear serum 

 removed to sterile ampules which arc sealed and labeled. Preserva- 

 tives should not be added as they tend to interfere with the preci- 

 pitin test. The serum should be perfectly clear and free from fat and 

 hemoglobin. It should be stored at about 4°C. If necessary the 

 serum may be filtered (Berkefeld). The titer of the precipitating 

 serum is determined by ascertaining the highest dilution of the anti- 

 gen with which the serum forms a precipitate or ring test in two 

 hours at 37°C. (optimum temperature). The precipitate consists 

 very largely of the globulin and lipids of the precipitating serum. 



PROCEDURE 



1. Progressively doubled serial dilulions of antigen are prepared in saline beginning 



with 1:100. (Tabled). 



2. Oue-tcntli ml. of the serum is transferred to the bottom of small tubes (5X50 mm.). 



3. An equal volume (0.1 ml.) of each dilution of antigen is layered onto the serum. 



4. Incubate at 37° for 2 hours and observe at 30 minute intervals for ring formation 



(precipitate at juncture of serum and antigen). 



5. Shake tubes and incubate overnight at 4°C. The precipitate will settle out and can 



be read by gentle shaking of the tubes. 



6. Controls of antigen with saline and serum with saline must be included and should 



show no precipitate. 



COMPLEMENT FIXATION 



The complement fixation test is based upon the observation that 

 the combination formed between an antigen and its specific antibody 

 has the property of uniting with complement. On the basis of this 

 general law, complement can be used to detect the union of an antigen 

 with its homologous or specific antibody. When a mixture of antigen 

 and antibody is furnished with an exactly sufficient quantity of com- 

 plement, all the complement is "fixed", or completely utilized in the 

 reaction and none is left free in the fluid to take part in any other 

 reaction between an antigen and its antibody which may be added 

 subsequently for test purposes. 



The test for such fixation is performed by placing together antigen, 

 antibody and complement in suitable proportions, as determined by 

 previous titrations, and subsequently testing for the disappearance 

 of complement. If the complement is not fixed, it indicates that the 

 antigen and antibody do not have the power to unite, or, in other 

 words, that the antigen and antibody are not specifically related. 

 On the other hand, the fixation of complement in the mixture indi- 

 cates that the antigen and antibody have combined, because of their 

 specific affinities. 



In some cases the union of the complement with the antigen-anti- 

 body complex produces a solution or lysis of the antigen. In other 



'See page VI1147-5-6. 



