576 IMMUNOLOGY 



1933) present evidence indicating that the exciting agents in the 

 pollens of ragweed, timothy and certain grasses are proteins, while 

 Black (1932) regards that of ragweed as a carbohydrate. 



Preparation of Pollen Extract. — To prepare a pollen extract 

 Rackemann (1931) recommends that one gram of pollen be added 

 to 100 c.c. of Coca's extracting fluid, the mixture shaken and 

 allowed to stand at room temperature for three days (it should 

 be shaken several times during this period). It is then filtered 

 first through paper and finally through a Berkefeld N filter. Ac- 

 cording to Rackemann, nitrogen determination shows that such an 

 extract contains approximately 0.20 mg. of nitrogen per cubic 

 centimeter while Coca (1934) says that a 1:100 extract contains 

 about 0.1 mg. of total N per cubic centimeter. The formula sug- 

 gested by Coca for an extracting fluid for pollen and house dust 

 is as follows : 



NaCl 0.5 per cent 



NaHCOg 0.275 per cent 



Phenol 0.4 per cent 



Carbon dioxide should be bubbled through this fluid until 

 phenolphthalein added to a sample of it remains colorless. The 

 formula for extracting fluids is varied according to the nature of 

 the material to be extracted. These formulas are discussed in de- 

 tail by Coca, Walzer and Thommen. 



Rackemann suggests that during pollen hay fever season fresh 

 extract should be made every two or three wrecks and kept sealed 

 in the ice box until ready for use. One must be certain of its 

 sterility. 



Standardizing Pollen Extracts. — Various methods of stand- 

 ardizing extracts have been employed. As previously mentioned, 

 Noon (1911) designated as a unit the amount of exciting agent 

 obtained by extracting one-millionth of a gram of pollen with 

 saline. Complement fixation has been employed, but is no longer 

 recommended. The reaction involves titrating antigenic fractions 

 of pollen against standardized immune serum obtained by im- 

 munizing suitable animals with pollen antigen. Since pollen pos- 

 sesses one or more antigenic fractions distinct from the exciting 

 agent of pollen hay fever, it is obvious that complement fixation is 

 a measure of antigenic fractions whose relationship to the exciting 



