HEMAGGLUTINATION BY ANIMAL VIRUSES 41 



4. Hydrogen Ion Concentration 



In general, hemagglutination occurs best at 4°C. and pH 7.0 with members 

 of group B, and at 37°C. and pH 5.6 to 6.4 with members of group A (Casals 

 and Brown, 1954); but there are large individual variations as, for example, 

 described for western equine (Chanock and Sabin, 1954a), West Nile (Chanock 

 and Sabin, 1954b), dengue (Sweet and Sabin, 1954), and St. Louis encephal- 

 itis viruses (Chanock and Sabin, 1953). 



Some hemagglutinins are unstable at the pH necessary to demonstrate 

 hemagglutination. It is then necessary to so buffer the cell suspension that 

 the pH of the virus suspension is adjusted suitably on addition of red cells 

 (Sabin, 1951). 



5. Relation of Hemagglutinin to Virus Particle 



It is generally assumed that the hemagglutinin is associated with the virus 

 particle. Sabin was able to deposit the hemagglutinin by centrifugation at 

 58,300 g (Sabin and Buescher, 1950), so that it is probably at least the size 

 of the infectious particle. 



No enzyme has been found associated with the hemagglutinin, and the 

 receptor-destroying enzyme of V. cholerae does not prevent adsorption of 

 the arthropod-borne viruses to red cells (Sabin and Buescher, 1950). 



6. Nonspecific Inhibition of Hemagglutinin 



Sera of normal humans, rabbits, monkeys, and mice contain an inhibitor 

 of the hemagglutinin. In human sera the inhibitor is active to a titer of about 

 1 : 1000 against 10 agglutinating doses of Japanese B virus (Sabin and 

 Buescher, 1950). 



Nearly all of the inhibitor was removed by extraction with chloroform, 

 benzine, acetone, or ether (Sabin, 1951), by Seitz nitration (Casals and Brown, 

 1954), or absorption with the clay Bentonite (Oker-Blom et al., 1950; Clarke 

 and Casals, 1955). Dialysis, filtration through sintered glass, or through 

 collodion membranes of APD, 303 nut, did not remove inhibitor, but mem- 

 branes of APD, 69m/x, retained part of the inhibitor from native serum, and 

 all the inhibitor from heated serum (Casals and Brown, 1954). Acetone ex- 

 traction is favored for the routine removal of inhibitor in practice, especially 

 with certain animal sera. 



The inhibitor was relatively stable to boiling and it was not affected by 

 proteinase or potassium periodate; but the lecithinase of Clostridium per- 

 fringens in the presence of calcium rendered it susceptible to heat inactivation 

 (Sabin, 1951 ; Sabin and Buescher, 1950). 



What is possibly a similar inhibitor is present in saline extracts of mouse 

 brain, from which it can be sedimented at 20,000 g for 60 minutes. If not 



