METHODS IN MAMMALIAN IMMUNOGENETTCS 369 



therefore not removed from the absorbed "reagent." The uncertainties of the system 

 probably increase exponentially with the number of components. Unless one is able 

 to purify his antigenic preparation to an essentially single component basis, therefore, 

 or unless he is working with an antigen that is certainly tagged, by something like 

 enzymatic activity, a unique absorption spectrum, or a tracer label that it does not 

 share with other molecules in the solution, or serologic specificities detectable by red- 

 cell tests, there is nowadays little justification for conducting immunogenetic work on 

 soluble antigens in mixtures by means of ordinary saline precipitation methods. The 

 simple precipitin system, however, has probably contributed more to our basic know- 

 ledge of serology than has any other. Readers are referred to discussions of this 

 system from a methodologic viewpoint, for example, by Kabat and Mayer 673 and 

 Boyd. 122 



Gel-diffusion serology. — A new era in the immunogenetics of soluble antigens was 

 ushered in by the development of gel diffusion methods by Oudin and Ouchterlony. 

 These methods make use of the diffusion of antigen or antibody or both into a gel 

 medium, usually agar. Since the different components of an antigenic mixture 

 generally diffuse at different rates, this technique permits the separation of com- 

 ponents of a mixture in a system comparable in some ways to chromatography. In 

 the Oudin method 974, 975 agar containing a known serum is allowed to solidify in a small 

 tube, and then an aqueous solution of the antigen is poured over the solid agar. As the 

 antigen diffuses into the underlying antibody gel, it forms a zone of precipitate for each 

 antigen-antibody pair present in adequate quantity. The Ouchterlony method is a 

 system of double diffusion ; the antigens diffuse through an intervening agar zone, to 

 meet a front of diffusing antibody. Where corresponding antibody and antigen meet, 

 a narrow line of precipitate is formed; and, when the antigenic preparation is a mixture, 

 a number of bands form, each corresponding to a particular antibody-antigen system 

 at positions related to the diffusion rates of the antigenic components and to the 

 relative concentrations of the antigens and antibodies. This system is subject to 

 quantitative analysis. 14, 349 



The method we use, adopted directly from G. J. Ridgway of the Bureau of Com- 

 mercial Fisheries, Seattle, is as follows. An agar base is prepared by mixing 1.5 per 

 cent Difco agar, 0.72 per cent NaCl, 0.6 per cent sodium citrate, and 0.01 per cent 

 Merthiolate, in distilled water. This solution is adjusted to pH 6.7 with HC1 before 

 the addition of trypan blue (to 0.01 per cent) while the agar is still hot. It is then 

 poured into test tubes, 8 ml. per tube. 



In setting up the tests, one tube of the hot agar base (kept in a boiling water bath) 

 is poured into a flat-bottomed petri plate. The plate should not be swirled to distri- 

 bute the agar. When the plate is cool, penicylinders (obtained from Fisher Scientific 

 Co.) are arranged in a pattern on the plate. We commonly use a porcelain peni- 

 cylinder at the center and stainless steel penicylinders arranged at the points of a 

 regular hexagon so that each is equidistant from the center. The distance from the 

 center of the central penicylinder to the center of each peripheral one is 2 cm. Shreffler, 



