NATURE AND FORMATION OF ANTIBODIES 21 



postulate of the template theory of antibody formation. During the past few 

 years we have attempted to prove it by chemical methods and to measure it 

 quantitatively. 



In our first experiments we used antigens labelled by large amounts of V^\ 

 of S^'-azophenylsulfonate or C'*-azobenzoate groups (Crampton and Hauro- 

 witz, 1950; Crampton, Reller and Haurowitz, 1952). We found that the radio- 

 activity of these substances persisted in the tissue proteins of the injected 

 rabbits for many months. Nine months after injection a liver or spleen cell 

 of the rabbit still contains an average of several hundred antigen molecules. 

 In rabbits injected with S^*-azo-proteins we found most of the protein-bound 

 S^^ in a fraction of the hydrolysate which, on chromatography, behaved like a 

 phenylsulfonic acid derivative (Haurowitz and Walter, 1955). Cystine and 

 methionine contained only a small fraction of the radioactivity. 



We have repeated these experiments with proteins containing S^*-amino 

 acids in their molecules. They were prepared by injecting animals with the 

 hydrolysate of yeast raised on S^^-sulfate. Some of these S^'^-proteins were then 

 coupled with I^''^ or with diazotized anthranilic acid containing C^^ These 

 doubly labelled proteins were used as antigens. We found again persistence of 

 the injected radioactivity. Indeed, the internal label, i.e., the label of the amino 

 acids, persists much longer than the externally attached I^^^ label, indicating 

 incorporation of S^^-amino acids or loss of fragments containing iodine (Fried- 

 berg, Walter and Haurowitz, 1955a; Idem, 1955b). When we injected proteins 

 internally labelled by S'^^ and externally labelled by diazotized C'*-anthranilic 

 acid, both labels persist in the organs and the ratio of S'^VC^'' increases much 

 less (Haurowitz, Ellenbogen and Walter, unpubl.). This indicates that many 

 of the antigen molecules persist as such. 



I see no reason, therefore, to invoke the formation of enzymes which form 

 antibodies in the absence of antigen, but I prefer to consider the antigen mole- 

 cule or its hapten as a template which persists for many months or years in 

 the organism and thus can give rise to the formation of many generations of 

 antibodies. This does not mean, however, that the antigen persists as such, 

 i.e., as easily soluble free antigen. We know from our own experiments that 

 considerable portions of the antigen are bound to the tissues and cannot be 

 extracted by isotonic saline solutions. Evidently, the antigen combines with 

 .some of the cellular constituents (nucleic acids, lipids, polysaccharides or other 

 , proteins) to form insoluble complexes; the antigen molecule in these complexes 

 is masked in such a manner that it does not react with added antibody (Hauro- 

 witz, Ellenbogen and Walter, unpubl.). 



The extent of complementariness can be estimated from the affinity between 

 antigen and antibody. One of the methods has been described by Dr. Press- 

 man. Another method can be applied when we are dealing with antigen-anti- 

 body precipitates. They can be washed repeatedly without losing too much of 



