274 MANUAL OF MICROBIOLOGICAL METHODS 



made to arrive at this end. Adsorption carried to the end of no function 

 of the antibody against a heterologous virus while retaining the function 

 for the homologous virus is of qualitative significance. It must be 

 remembered, however, that the procedure described above, while effective 

 in removing antibody for the available antigens of the virus particle, 

 may not have removed antibody for soluble antigens, depending on the 

 extent of aggregation of the antigen-antibody molecules and perhaps on 

 other factors. Although such soluble antigens may be presumed to be 

 bound to their antibodies, the particle-adsorbed serum should be used 

 only with the awareness that the soluble antigen-antibody system is 

 probably subject to the same factors affecting other antigen-antibody 

 systems. 



Receptor -destroying Enzyme 



It would be amiss to attempt a prolonged discussion of inhibitors in 

 this technical section. Some consideration of inhibitors, however, 

 cannot be omitted from even the most basic discussion of technic. One 

 can do almost no virological investigation without using reference anti- 

 serum to be certain of his virus. Antiserum is one source of inhibitor. 

 The presence of an inhibitor in antiserum used in hemagglutination- 

 inhibition studies may be the cause of false conclusions. If the inhibitor 

 titer is greater than the antibody titer, one cannot expect to read the 

 antibody function accurately. A case in point would be the parallel 

 titration of acute and convalescent serum. If the convalescent serum 

 possessed a relatively low titer of antibody and the acute serum possessed 

 a relatively high titer of inhibitor, a rise in antibody would be over- 

 looked. Since the inhibitor can cause results comparable to antibody 

 in the hemagglutination-inhibition test, it is best removed. 



Certain strains of Vibrio cholera, e.g., 4-z, elaborate an enzyme capable 

 of inactivating the inhibitor in question. To prepare cholera receptor- 

 destroying enzyme (RDE) a young agar slant culture of the vibrio is 

 used to inoculate a shallow layer of brain-heart-infusion broth, 1.5- 

 2.0 cm deep, in a large flask. The culture is incubated 24 hr at room 

 temperature (26-28°C), and most of the bacteria are removed by cen- 

 trifugation. The remainder are removed by filtration through a Seitz 

 pad, and the filtrate is checked for sterility and activity. The activity 

 may be checked against chicken red cells or against serum inhibitor. If 

 it is to be used to remove serum inhibitor, it probably would be better 

 to use the latter substrate. The enzyme apparently loses its activity 

 slowly at 4°C, for it is usable for several weeks if so stored. 



To treat a serum with unpurified enzyme, 1 part of serum is added to 

 1 part of filtrate and the mixture is incubated 18 hr at 37°C. The ratio 

 of serum to enzyme will vary somewhat with the potency of the enzyme. 



