VIROLOGICAL METHODS 275 



The enzyme is then inactivated at 62°C for 30 min. The treated sample 

 and a sample of untreated serum are titrated under the same conditions 

 to demonstrate the change in inhibitor titer. The dilution caused by 

 the addition of the enzyme, of course, must not be overlooked. If the 

 enzyme is not inactivated prior to the hemagglutination-inhibition test, 

 it is free to act on the red-cell receptors, thus equivocating the whole test. 

 Receptor-destroying enzyme requires calcium ion, but its activity is 

 impaired by citrate ion. The formula for buffered calcium saline follows: 



Grams 



Calcium chloride (CaCh) 1 .000 



Sodium chloride (NaCh) 8 . 500 



Boric acid (H3BO3) 1 . 203 



Sodium tetraborate (Na2B4O7-10H2O) 0.052 



Distilled water 1,000 . 000 



The enzyme preparation can be serially diluted in the calcium saline 

 when its activity is assayed against red cells. If the concentration of 

 the red cells and the incubation time and temperature are maintained 

 constant, the potency of the enzyme can be determined by extinction 

 dilution. The end point is described as the least amount of enzyme 

 capable of rendering the red cells inagglutinable by the virus in question 

 under standardized conditions. It is advisable that the worker consult 

 original articles to determine further activities of the receptor-destroying 

 enzyme (Burnet et al., 1946). 



COMPLEMENT FIXATION 



Basic Technic 



The basic technics employed in complement-fixation tests have been 

 described in Chap. IX. It would seem advisable to comment only 

 on the aspects of the test more troublesome or peculiar to virology. 



Antigens. Since we are unable at this time to propagate large quanti- 

 ties of viruses on artificial culture media, obtaining a strong antigen is 

 one of the more common problems. Freeing viral antigens from host- 

 tissue elements which may be anticomplementary or be otherwise objec- 

 tionable not uncommonly presents another problem. 



The viral antigen functioning in the complement-fixation test may be 

 intimately associated with the viral particle, soluble and separate from 

 it, or may be found under both circumstances. During an active infec- 

 tion of host cells it may increase simultaneously with or independent 

 from other virus propensities. 



The preparation of antigen varies considerably with the virus and/or 

 tissue in which the virus was propagated. Antigens from allantoic or 



