22 FELIX HAUROWITZ 



their weight. Using precipitates in which either the antigen or the antibody 

 was radioactive, we found that only antibody is removed by washing and that 

 the so-called solubility of the precipitates is not true solubility but loss of 

 antibody (Haurowitz, Crampton and Sowiski, 1951). In the experiments in 

 which radioactive antibody was used, it became possible to measure the very 

 low concentrations of antibody in the washings and to estimate the apparent 

 association constants of the antigen-antibody complex. This constant is called 

 apparent because it increases on repeated washing from an initial value of 

 approximately 10^ to values of about 10* and more, thus indicating that the 

 precipitate first gives off loosely, later firmly bound antibody molecules. The 

 difference in affinity may be caused either by sterical factors which affect the 

 combination of antigen with antibody or by differences in complementariness. 

 That such differences exist, that each immune serum contains a variety of 

 well and poorly adapted antibodies is quite clear from serological experiments 

 (Haurowitz, 1942). 



How many different types of antibodies can an animal form? At first sight 

 this seems to be an unanswerable question. However, if we investigate the 

 specificity of antibodies formed in response to antigens which carry different 

 determinant groups, A and B, separated from each other by more than about 

 15 A, we find that either anti-A or anti-B is formed, but not anti-AB; evidently, 

 the determinant area of an antigen molecule is not larger than about 100-200 

 A-. If the antigen is a foreign protein, this area cannot contain more than 

 about 3 or 4 amino acid residues. We know at present of 16 types of amino 

 acids occurring in most proteins. A simple calculation shows that there are 

 about 5000 possible permutations of 3 and about 60,000 permutations of 4 

 amino acid residues. Many of these permutations may not occur at all in na- 

 ture; others may not be serologically active. There may not be more than a 

 few thousand serologically active permutations of 3 or 4 amino acids; accord- 

 ingly, the number of possible antibodies against proteins may not exceed a 

 few thousand. This agrees with the well known fact that antibodies against 

 proteins of closely related species such as hen and duck show serological cross- 

 reactions. Likewise, antibodies against the carbohydrate of blood-group A 

 combine with the polysaccharide of pneumococcus type XIV (Goebel et al, 

 1939). Evidently, the number of antibody types which can be formed by an 

 animal is not indefinite but of the order of 10^ to 10^. 



Determining the amount of injected antigen and of circulating antibody, 

 we can calculate the number of antibody molecules formed per injected antigen 

 molecule and the maximum time required for the formation of an antibody 

 molecule. As a basis for such a calculation the figures of Cohn and Pappen- 

 heimer (1949) can be used; these authors, after injection of a single dose of 

 diphtheria toxoid, observed formation of approximately 2 g. of antibody per 

 microgram of injected toxoid during a period of three weeks. Taking into con- 



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