VIROLOGICAL METHODS 271 



virus causing a complete positive is considered to be the end point, and 

 the final dilution of the virus fluid in this tube is the hemagglutinin titer. 

 Since the relationship of hemagglutinin to virus mass or particle 

 number is not accurately known either between viruses of different 

 antigenic types causing the same disease or between viruses causing 

 different diseases, one should be cautious about quantitative conclusions 

 along this line. 



SEROLOGICAL PROCEDURES 



Antihemagglutination or Hemagglutination Inhibition 



As with a number of other viral functions hemagglutination can be 

 prevented by specific antibody. This inhibition is not to be confused 

 with inhibition of hemagglutination caused by substances other than 

 antibody and discussed elsewhere. A certain minimal amount of anti- 

 body is required to prevent hemagglutination caused by a specified 

 amount of virus under standard conditions. Therefore, quantitative 

 antibody estimations can be made under appropriate conditions such 

 as the difference in antibody titers of acute and convalescent sera from a 

 given patient against a given sample of virus. 



The same materials are used as for the hemagglutination test except, 

 of course, a specimen of serum is also required. The serum may require 

 preliminary centrifugation to render it free from particulate matter. 

 Ten tubes are arranged in the first row, and seven in the back. Nine- 

 tenths milliliter of saline is pipetted into tube 1, row 1; 0.25 ml to the 

 remaining nine tubes in the same row; 0.5 ml to tubes 1-5, inclusive, 

 and tube 7 in row 2; and 0.25 to tube 6. When sera suspected of having 

 a high antibody is used, a preliminary dilution of 1:10 may be necessary. 

 Otherwise 0.1 ml of serum is added to tube 1, row 1. After the contents 

 of this tube are mixed, 0.75 ml is withdrawn, 0.25 ml discarded, 0.25 ml 

 added to tube 6, row 2, and the final 0.25 ml used to continue the serial 

 dilution in tube 2 and remaining tubes in row 1. Discard the final 

 0.25 from tube 10. 



The virus concentration used in the test is held constant. The 

 hemagglutinin titer of a given sample of virus having been determined, 

 the sample is diluted in saline to contain 16 hemagglutination doses per 

 millihter. Virus so diluted should be used immediately. Add 0.25 ml 

 of the diluted virus to each of the 10 tubes in row 1. Pipet 0.5 ml to 

 tube 1, row 2, and serially dilute through tube 5 by carrying 0.5 ml. 

 Discard the 0.5 ml from tube 5. Shake the rack to mix the ingredients 

 well, and after a brief waiting period (5 min) pipet 0.5 ml of 0.25 per cent 

 red cells to each of the 16 tubes. Shake the rack again, and allow it to 



