214 MANUAL OF MICROBIOLOGICAL METHODS 



placed in a small test tube. The other reagent (antigen) is layered over 

 the agar, and the test allowed to incubate 4 to 7 days. If only one 

 antigen is present, there will be only one zone of precipitation, but if addi- 

 tional antigens are present, additional rings will be seen, assuming, of 

 course, that the serum contained antibodies directed against the other 

 antigens and also assuming that two rings do not coincide because of 

 diffusion rates and concentration. 



In the double-diffusion procedure, agar which contains neither reactant 

 is poured into a petri dish. The antigen and antibody are placed in wells 

 and allowed to diffuse toward each other with the resultant formation of 

 lines of precipitation. Since a complete discussion of these procedures is 

 available in the review by Oudin (1952), the details have been omitted 

 here. 



The quantitative measurement of precipitin. The procedures described 

 elsewhere in this section are entirely adequate for some applications 

 involving microbial antigens. In other instances, it may be more profit- 

 able to employ a technic that permits the precise estimation of antibody 

 contained in a precipitate. This may be done with a variation of the 

 quantitative precipitin test as developed by Heidelberger and Kendall 

 (1929, 1933, 1935). 



When microbial antigens are being studied, the quantitative precipitin 

 test is useful for closely related (cross reacting) antigens, for studjdng the 

 contamination of one antigen by another, for estimating concentration 

 of an antigen present in a solution, or for any other application where the 

 less quantitative technics are insufficiently sensitive. 



In the usual form of the quantitative test, increasing amounts of anti- 

 gen are added to constant volumes of antiserum and the tubes stored at 

 some constant temperature (0-4°C) for several days until equilibrium 

 has been reached. The precipitates are then carefully removed by 

 centrifugation, and extraneous serum proteins are removed by rinsing 

 the precipitates with ice-cold sahne solution. The precipitates are 

 analyzed for nitrogen by a micro-Kjeldahl procedure. In the hands 

 of a careful investigator this method permits accuracy that is comparable 

 to that obtained in quantitative analytical chemistry. In addition to 

 nitrogen analysis, antibody has been measured quantitatively in other 

 ways. A procedure that is useful for serums with low concentrations of 

 antibody is the Folin Ciocalteau analysis for tyrosine (Heidelberger and 

 MacPherson, 1943). 



While the quantitative estimation of precipitins is a relatively simple 

 operation, its proper evaluation may require more information than it is 

 possible to impart in a chapter of this magnitude. Since other sources of 

 material (Kabat and Mayer, 1948; Cohn, 1952) are available, further 

 description of the procedures has not been included here. 



