QUANTITATIVE TECHNIQUES IN ANTIGENIC ANALYSIS 275 



corresponding antibodies in the serum, and that the various antibodies in the serum reflect 

 the various antigens in the bacterium. 



. We have seen on p. 251 that certain complex antigens appear to possess more than 

 one kind of determinant, and stimulate the production of an antibody Avith two or more 

 specificities (see Bumet 1934, Heidelberger and Kendall 1934, Morgan 1936a, Meyer 1938, 

 1939, Miles 1939). If then we postulate a bacterium x with an antigenic complex ab, 

 and a second bacterium y with an antigen a, the results of absorption tests would indicate 

 that z and y were antigenically identical, since ab would remove all A agglutinins from 

 the anti-y serum and a would remove the AB agglutinins from the anti-:c serum. Indeed, 

 the only way we should ever discover the existence of the complex ab would be in com- 

 parison with two other bacterial species, which happen respectively to possess a only and 

 b only. If, on the other hand, we postulate that the response to bacterium x (with the 

 complex ab) consists of separate antibodies A and B, then absorption tests would lead 

 us to postulate that x and y had an antigen in common, and that x had in addition a 

 distinct antigen pecuhar to itself. The extraction and separation of the various antigens 

 provides a way out of this impasse. The different antibodies may then be removed from 

 the bacterial antisera in specific precipitates, and the precise specificity of the remaining 

 antibodies for the other antigens determined. 



Quantitative Techniques in Antigenic Analysis. 



The methods of antigenic analysis discussed above yield qualitative answers. 

 Estimation of the relative amounts of the various antigens is possible only if certain 

 assumptions are made. These are, that there is a quantitative correspondence 

 between the different kinds of antibodies in a serum and the different kinds of 

 antigens present in the homologous bacterium ; that all the antigens have the 

 same antigenic capacity, weight for weight, and that each of the antigens reacts 

 with its corresponding antibody with the same degree of intensity. None of these 

 assumptions is warranted, and they are indeed seldom made explicitly, though 

 they are sometimes implicit in the inferences drawn from analytical data. It will 

 be clear from Chapter 7 that the last two assumptions are unjustified. The first 

 requires a little amplification. 



If a bacterium y removes half the antibody from anti-ic serum, the fact that 

 a large amount of antibody peculiar to x remains is sometimes interpreted as 

 indicating a large amount of corresponding antigen in x. But, as Miles (1939) 

 pointed out, the antibody response to many repeated doses of a small amount of 

 antigen may be as great as that to many doses of a large amount, so that after 

 prolonged immunization with a bacterium containing a major and a minor antigen, 

 the corresponding antibodies may be in equal concentration in the serum. Kemoval 

 of the antibody to the major antigen would in this case leave a large amount of 

 antibody representing what was in fact a minor antigen. 



Nevertheless, though quantitative correspondence of antibody and antigens is 

 unlikely ever to occur, it is clearly important to know how much of the various 

 antigens and antibodies are contained in the two reagents, a bacterial suspension 

 and an antiserum, at our disposal. A knowledge of the antibody concentrations is 

 particularly useful. For example, the quantitative relationship in antigen-antibody 

 reactions described in Chapter 7 permits the measurement of the concentration 

 of one antigen in a mixture of antigens derived from a bacterium. 



Heidelberger and his colleagues (see Heidelberger and Kabat 1934, 1936, 1937, 

 1938, Alexander and Heidelberger 1940, Henriksen and Heidelberger 1941) have 

 measured specific agglutinin content in terms of maximum antibody nitrogen 

 adsorbed by known doses of bacterial suspensions. The data so obtained permit 



