SEROLOGICAL METHODS 209 



a chapter of this length. Since the procedure is well presented in Kabat 

 and Mayer (1948), further description has not been included here. 



PRECIPITATION 



When a clear solution of a protein or polysaccharide antigen is mixed 

 with the appropriate antibody or precipitin, the mixture turns cloudy and 

 then precipitates. This reaction differs in principle from agglutination 

 chiefly in the size of the particles involved. In agglutination, the par- 

 ticles are usually cells and in any event are large enough to be seen under 

 the microscope, while in the precipitin reaction, the particles are of 

 molecular dimensions. 



Preparation of reagents. It is essential that both antigen and antibody 

 solution be perfectly clear and free from lipid or insoluble material. 

 Lipid in serum can usually be avoided by withholding food from the 

 animal for 24 hr before bleeding. If this precaution has not been taken, 

 lipid can be removed by storing the serum at 0°C for 3-5 days and then 

 centrifuging at 2,000-5,000 X gravity to bring lipid to the surface. Any 

 particulate matter other than lipid will be thrown to the bottom of the 

 centrifuge tube, and the clear serum may be withdrawn with a capillary 

 pipet. Clarification can also be accomplished by passing the chilled 

 serum through a precooled Seitz filter containing a clarifying pad (average 

 pore diameter 5 m) or a coarse sterilizing pad (average pore diameter 1 ju). 



Antigens to be used for qualitative precipitin tests may consist of exu- 

 date from an infected animal or cell-free fluid from a broth culture. In 

 other instances, bacteria may be disrupted by mechanical grinding or by 

 sonic oscillation and the soluble products used as antigen. 



For precise work, it mil be necessary to purify the antigen under study 

 by the physical and chemical procedures customarily employed for pro- 

 teins, polysaccharides, and other high-molecular-weight compounds. 



Concentration of antigen is usually expressed in terms of weight in 

 milligrams or micrograms per milliliter of solvent when dealing with puri- 

 fied antigens. In the case of protein or other nitrogen-containing anti- 

 gens, concentration may be expressed as milligrams or micrograms of 

 nitrogen based on a micro-Kjeldahl analysis. For crude antigens like 

 cultural supernates or lysates, concentration is ordinarily expressed in 

 terms of dilution of the original cultural fluid. This, of course, is less 

 desirable than expression on a weight basis but is satisfactory for applica- 

 tions where strictly quantitative results are unnecessary. 



The electrolyte concentration should be maintained within certain 

 limits. With most systems, a small amount of electrolyte is necessary 

 for precipitation, and if a great excess is present, precipitation wiU be 



