204 MANUAL OF MICROBIOLOGICAL METHODS 



tion, mixed with water, and dialyzed against cold running water for 

 2 days. Dialysis can be hastened by placing the cellophane dialysis 

 casing containing the globulin into a large carboy partially filled with 

 saline and allowing the carboy to rotate in a horizontal position on a ball- 

 mill assembly. This operation should be conducted in the cold, and the 

 saline should be changed frequently. 



Highly purified antibody for pneumococcus polysaccharide has been 

 prepared by Heidelberger and his coworkers by dissociating the antibody 

 from specific precipitates or agglutinates with 15 per cent sodium chloride 

 solution or with barium hydroxide and barium chloride (see Kabat and 

 Mayer, 1948). 



The low-temperature ethanol-precipitation methods devised by Cohn 

 and his coworkers are also useful for isolating gamma globulin and 

 thereby freeing antibody of much extraneous material present in crude 

 serum (Deutsch, 1952). 



Various forms of electrophoresis apparatus can also be used for iso- 

 lating gamma globulins or other globulins with antibody activity. Per- 

 haps the best suited to serum fractionation is the electrophoresis-convec- 

 tion apparatus of Kirkwood (Cann et al., 1949). An application of this 

 procedure to fractionation of rabbit antibody may be found in the paper 

 of Cann et al. (1951). Zone electrophoresis (Flodin and Porath, 1954) 

 and continuous-flow paper electrophoresis (Durrum, 1951) would also 

 seem to have merit for some applications. 



Antibodies against protein antigens have been purified by a novel pro- 

 cedure (Sternberger and Pressman, 1950). The protein antigen was 

 first coupled to p-aminobenzenearsonic acid or o-aminobenzoic acid 

 through a diazo linkage. Following this, the coupled antigen was pre- 

 cipitated with antibody. The precipitate was washed and dissolved in 

 saturated calcium hydroxide solution. Calcium aluminate was used to 

 precipitate out the antigen, leaving the pure antibody in the supernate. 



Isliker (1953) has purified isohemagglutinin by coupling the blood- 

 group antigen to an ion-exchange resin. The antibody was allowed to 

 bind to the antigen-resin complex and subsequently eluted by a change 

 in pH or by simple sugars. 



Antibody absorption. Antibody prepared against one microorganism 

 often cross-reacts with other microorganisms that are taxonomically 

 related, and less commonly the antibody may cross-react with distantly 

 related organisms or even higher plants and animals. Since microbial 

 cells are rather complex mixtures of antigenic substances, a cross reaction 

 may be due to the sharing of one or more common antigens. On the 

 other hand, it sometimes happens that cross reactions occur between two 

 purified antigens that are known to be chemically homogeneous. In this 

 case the cross reaction cannot be due to shared antigens but is due to 



