UNION OF ANTIBODY WITH ANTIGEN 



121 



the negative group are fully ionized. A similar relation would apply 

 in the alkaline region. If two negative and two positive groups were 

 critically involved in each Ab-Ag bond, the expected relation would 

 now contain a (pH)- and a 2(pH) term. 



Singer tested this relation by ultracentrifugal observations on Ab- 

 Ag mixtures at different pH. Typical results are shown in Fig. 9-3, 



Fig. 9-3. Ultracentrifugal diagrams of mixtures of bovine serum albumin 

 and its antibody at various pH. Sedimentation is proceeding in the direction of 

 the arrow. Ag stands for antigen, Ab for antibody, a for an antibody-antigen 

 complex thought to be AgoAb, b represents a complex thought to be AgsAb-, 

 and 5 is gamma globulin. At pH less than 4.5, progressively larger amounts of 

 free gamma globulin (antibody) appear, while the amounts of the complexes 

 diminish (Singer, 1957). 



which shows the sedimentation diagrams of mixtures of bovine 

 serum albumin and rabbit anti-bovine serum albumin. As the pH 

 falls, more and more free gamma globulin (antibody) appears in the 

 mixture while the amount of the antibody-antigen complexes de- 

 creases. The changes are clearly a function of pH* and were fomid 

 to be entirely reversible. 



Enough results at different pH were obtained to show that the 

 linear relation predicted by the equation holds quite well (Fig. 9-4). 

 This was found to be true for both systems studied, namely, oval- 

 bumin-antiovalbumin and bovine serum albumin-antibovine-serum- 



* Habeeb et al. (1959), however, conclude from chemical modification studies 

 that "the removal of the positive charge on the same amino groups of Ab by 

 an increase of the pH of the solution, instead of by acetylation, might have 

 the same effect on the Ab molecule and its capacity to precipitate witli a 

 large Ag molecule. The generally-observed dissociation of Ag-Ab bonds in 

 alkaline solution might therefore be attributable to such a deformation of the 

 Ab molecule, rather than ... to titration of specific critical groups within the 

 Ab sites." 



