Immunological approaches to the study 

 of ribonuclease 



B.CINADER AND J. H. PEARCE* 



Agricultural Research Council, Institute of Animal Physiology 

 Babraham, Cambridge 



IMMUNOLOGICAL CRITERIA OF HOMOGENEITY 



The immunological analysis of a protein provides one of the most sensitive 

 methods for the detection of macromolecular impurities. Diffusion and pre- 

 cipitation of antigen and antibody in agar (Oudin, 1952) leads to discrete 

 precipitin zones for each antigen, the total number of independent zones 

 representing a minimum estimate of the number of antigens present. 



Since individual animals vary in their responsiveness to the same antigen 

 (Sobey, 1954) and since a massive antibody response occurs more readily 

 to some antigens than to others, it is necessary to examine diffusion and 

 precipitation in agar with antisera from several individuals and with the 

 antisera from the same animals after successive courses of immunization. 



Molecules which are indistinguishable as antigens (Heidelberger, 1954) 

 but are of different mobility can be distinguished by electrophoresis in agar 

 and by their subsequent reaction with antibody diffusing from channels 

 placed parallel to the direction of electrophoretic migration (Williams and 

 Grabar, 1953; see also Cinader and Dubert, 1955). 



The assay of the nitrogen contents of precipitates of antigen and antibody 

 gives not only information on the combining ratio of reactants, but also 

 on the relative purity of different preparations of the same protein. The 

 shape of a plot of total N precipitated as a function of antigen N added 

 to a constant volume of immune serum (Cohn, 1952) and the quantity of 

 antigen leading to maximum precipitation of antibody (Cinader and Pearce, 

 1956) can serve as a sensitive index of relative purity. 



The supernatants from antigen-antibody precipitates may provide addi- 

 tional information on the heterogeneity of the antigen. The simultaneous 

 presence of antigen and antibody in the same supernatant has usually been 

 accepted as indicating the presence of antigenic contaminants. However, 

 this is not necessarily so ; only the examination of supernatants by diffusion 

 and precipitation in agar can show whether the presence of antigen and 

 antibody in the same supernatants is due to soluble complexes of the prin- 

 cipal antigen and its antibody or to antigenic contaminants. 



* Present address : Microbiological Research Establishment, Porton, Wiltshire. 



