Small amounts of formaldehyde inhibited the precipitating activity of horse diphtheria antitoxin with toxin and of horse antipneumococcus serum with the homologous capsular carbohydrate. Approximately 1 part of commercial formaldehyde to 1000 parts of serum, acting for 24 hours, inhibited the flocculating activity completely. In both cases, the combining affinity of the treated antibody for the corresponding antigen was not demonstrably affected, as determined both by in vitro experiments and by animal protection. More intensive treatment of the antipneumococcus serum caused an apparent loss of its bacterial agglutinating activity, but on centrifugation the organisms cohered: combination had occurred, and only the spontaneous aggregation was prevented. These effects are the same as those previously described for diazo compounds, and have been qualitatively reproduced with acetaldehyde, benzaldehyde and butyraldehyde.
The quantitative relationships suggest that only a few groups in the antibody molecule need be modified by formaldehyde in order to prevent aggregation; and it is probable that these are some of the free NH2 groups of the antibody protein. In marked contrast, the combining affinity of both antipneumococcus antibody and diphtheria antitoxin for the corresponding antigens was only slightly affected by amounts of formaldehyde which sufficed to block the free NH2 groups rapidly and almost completely. Similarly, this amount of treatment did not affect the reactivity of these two antisera acting as antigen with a rabbit antiserum versus horse serum. The integrity of the NH2 groups is apparently not essential for the activity of these sera acting either as antigen or as antibody; and the slow disappearance of their activity in concentrated HCOH is apparently to be ascribed to some secondary reaction other than the simple addition of HCOH to free NH2 groups.
The present experiments do not support the theory that antigen-antibody aggregates are lattice-like structures built up from elementary antigen-antibody compounds because of residual specific combining groups. The aggregating activity of both antipneumococcus serum and diphtheria antitoxin was completely inhibited by procedures which did not demonstrably affect their combining power with antigen. This suggests that the aggregation of antigen-antibody compounds is a secondary, non-specific reaction. It is perhaps significant that the amount of formaldehyde which just sufficed to prevent aggregation also caused a marked increase in the solubility of the pneumococcus antibody, which could then no longer be precipitated at serum pH by dilution with water or by dialysis. This strongly suggests that the loss of precipitating activity is actually due to the increased solubility of the antibody and supports the hypothesis that the primary cause of specific antigen-antibody aggregation is the relative insolubility of the bound antibody.