370 IMMUNOGENETICS 



in our laboratory, has prepared lucite guides which fit on the petri plate, with holes 

 bored in the proper position just large enough to drop the penicylinders through to the 

 agar surface. After the penicylinders are in position, another tube of 8 ml. of the hot 

 agar base is poured around them. Again, the plates should not be swirled. 



The antiserum is then placed in the central penicylinder, filling it, and the test 

 antigens are placed in sequence in the peripheral ones. Plates are sealed with rubber 

 tape and placed at 37° C. for about a week (the time necessary will vary with the anti- 

 gens and the serum) . The precipitin lines that form are then photographed ; a modified 

 dark-field type of illuminator producing good photographs has been described by 

 Klontz et al. 72e The trypan blue in the agar, and the porcelain central penicylinder, 

 improve the quality of photographs. 



Numerous variations of both the Oudin and Ouchterlony procedures have been 

 described. Preer 1021 has described a method adaptable to the use of very small 

 quantities of material. Many investigators prefer to use molds of various design and 

 to pour the agar medium for gel diffusion tests around the molds. When the molds 

 are removed, wells remain in the solidified agar. Sets of agar-gel cutters for various 

 purposes, together with descriptions of their uses and references, can be obtained from 

 Shandon Scientific Co. Ltd., 6 Cromwell Place, London. 



Gel-diffusion methods have made possible the first extensive and definitive work 

 with the immunogenetics of mammalian soluble antigens in precipitating systems, for 

 example, genetically controlled variations in the specificity of rabbit serum globu- 

 lins 974, 975 and the sharp inherited differences in the quantity of a serum protein 

 distinguishing particular inbred lines of mice. 



Immunoelectrophoresis. — Another powerful technique for dealing with the serology 

 of mixtures has been developed mainly by Grabar 472 and extended by many others. 

 Essentially, the procedure first subjects the antigenic mixture to electrophoresis. 

 After the electrical field has separated the components of the mixture along a linear axis 

 as a function of their charge at the particular pH and ionic strength of the medium, the 

 components of the mixture are permitted to diffuse through the agar to meet a front of 

 diffusing antibody, so that a band of precipitate forms where antigens and their corre- 

 sponding antibodies meet. The preliminary electrophoretic step provides an additional 

 dimension of separation of the antigenic components ; the technique has been proved 

 especially effective for the discernment of many components in very complex mixtures, 

 such as whole serum or plasma. Bussard 150 has described a useful modification, in 

 which the precipitation occurs during the course of electrophoretic migration. 



A combination of starch-gel electrophoresis with gel-diffusion serology has been 

 reported by Schwartz. 1178 Starch gels, after the method of Smithies, 1229 generally 

 make cleaner electrophoretic separations than do agar gels, and the addition of anti- 

 body precipitation to this system is a powerful technique indeed. 



Complement fixation. — A classical serologic procedure of high sensitivity and specifi- 

 city is the system of complement fixation. In principle, the system is simple: an 

 antigen is allowed to react with its corresponding antibody in the presence of comple- 



