Sufficient coupling with any of five different diazo compounds eventually destroyed the reactivity of all the antisera here studied. The rates of inactivation varied considerably among the several antisera. By stopping the reaction at intervals, it was possible to prepare partially inactivated antibodies of peculiarly modified reactivity.
Thus, the flocculating activity of diphtheria antitoxin with toxin was completely destroyed long before there was any demonstrable impairment of its protective titer in vivo. The first change induced in antipneumococcus horse sera was the apparently complete loss of reactivity with the capsular carbohydrate at a time when the agglutinating, animal-protecting and complement-fixing activity of the sera were only slightly affected. On further coupling, the sera no longer caused visible agglutination; but aggregation of the serum-treated bacteria could be induced by centrifugation. Still further coupling destroyed all antibody activity.
Rabbit antisera to egg albumen and horse serum no longer precipitated the homologous antigen after treatment with diazo compounds, probably due to their failure to combine with the antigen. The hemolytic, complement-fixing and lipoid-flocculating activity of coupled rabbit antisera to sheep red blood cells fell off in parallel; the hemagglutinin seemed somewhat more resistant. The reagin of syphilitic serum was destroyed almost instantaneously by comparatively small amounts of diazo compounds. Finally, in the case of antityphoid agglutinin, the isoelectric point of the coupled antibody, measured on the surface of specifically sensitized bacteria, was found to shift from an original value of pH 4.7 to one of less than pH 2.7 as progressively more sulfanilic acid radicals added on to the antibody molecule.
The groups in protein which participate in its reaction with diazo compounds probably include aliphatic amines, the imidazole ring of histidine, the indole group of tryptophane, the NH of proline and hydroxyproline and the phenyl group of tyrosine. Although it has been possible to modify antibodies chemically so that they combine with the corresponding antigens without causing their aggregation, the experiments here described furnish no indication as to which of these groups in antibody protein are primarily concerned in the antigen-antibody reaction, and which are responsible for the secondary flocculation. Such localization awaits the development of a technic for attacking individual groups in the protein molecule.