INTERACTIONS OF HAPTENS WITH ANTIBODIES 275 



The basic procedure is to produce antibodies in rabbits by injecting sheep 

 serum coupled with some substance such as diazotized p-arsanilic acid and 

 then precipitate these antibodies by mixing with simple polyhaptenic sub- 

 stances possessing 2>-diazophenylarsonate groups. When various substituted 

 monohaptenic phenylarsonates are present, the precipitation is inhibited 

 due to these substances competing with the polyhaptens for the specific 

 binding sites. From the degree of inhibition produced, the binding relative 

 to a standard hapten can be determined and expressed as a combining 

 constant K^; the standard hapten, such as phenylarsonate, is assigned a 

 Kq = 1. These constants then are not absolute and do not relate directly 

 to the free energy of binding. However, from them may be calculated the 

 difference in binding energy between any two compounds using Eq. 6-105 

 (with a numerical factor of 1.273 since equilibrium is usually at 5°). It is 

 usually convenient to compare the substituted phenylarsonate haptens 

 with the standard unsubstituted compound, in which case the energy dif- 

 ferences may be correlated with the substituent group. 



protein — N=N— <f >— AsOgH" 

 Antigen 



_ OH OH 



-H03As-<^_^-N=N-^^-N=N-|^\/^-N=N-^\-N=N-^^\-As03H- 



HOgS-^ys^^-SOaH 

 Polyhapteni c precipitant 



C >-As03H~ 

 Standard inhibitor 



X-/ \-AsO3H- 

 Test inhibitor 



Determination of Group Interaction Energies 



The binding of the inhibitor hapten X— <p — AsOgH^ to the antibody pro- 

 tein would result from the following interactions: (1) the electrostatic at- 

 traction between the negative arsonate group and a positive charge on the 

 protein; (2) dipole-dipole interactions between the arsonate group and the 

 protein; (3) the dispersion attraction between the arsonate group and the 

 benzene ring for the protein; (4) the dispersion attraction between the 

 group X and the protein; and (5) other interactions between the group X 

 and the protein, depending on the nature of X. Comparing the inhibitor 



