The Effect of the Order of Addition of Antigen and Antibody on the Precipitin Reaction

Abstract

The existence of the “order of addition effect”, that less precipitate is obtained when antiserum is added to antigen than when antigen is added to antibody, has been demonstrated for both rabbit and horse antisera. The fundamental identity of this finding to the Danysz phenomenon was pointed out. The explanation offered previously for the Danysz phenomenon, the disparity between the speed with which the initial reactions of antigen and antibody occur, and the slowness with which certain of the subsequent reactions take place, has been accepted for the “order of addition effect”. The presence of the order effect was used as a measure of the lack of equilibrium in the precipitin reaction. With rabbit antisera it was revealed that under the usual conditions of incubation and refrigeration equilibrium was not attained in either antibody excess or antigen excess. With the horse antiserum studied, in confirmation of the work of Gitlin and Edelhoch, equilibrium was approached speedily in antigen excess, but much more slowly in antibody excess. The removal of complement by treatment of the antiserum with versene or immune precipitate tended to diminish the order effect with some sera, but was without effect on others. In contradiction to the findings of Maurer and Weigle, versene tended to decrease the amount of precipitate formed with most rabbit antisera studied. The possible significance of these findings to the mechanism of versene action was briefly indicated. It was concluded that, as suggested by Kabat and Schorr, complement tends to decrease the velocity of certain of the antigen-antibody reactions. With the horse antiserum studied versene was found to increase the amount of precipitate obtained in antibody excess, although a decrease was found in antigen excess and equivalence.