The Effect of Unheated Normal Bovine Serum on The Complement Fixing Activity of Heat Inactivated Bovine Antiserum With Homologous Antigen: II. Aggregative Properties.

To determine whether the greater fixation of complement observed in "modified" complement-fixation tests is related to an increased aggregation of the antigen-antibody complexes, parallel tests by the two methods have been made with two different particulate bacterial antigens and corresponding bovine antisera. At the end of the fixation period the mixtures were centrifuged, the supernatant fluids removed carefully, the sediments washed twice and re-suspended in a small volume of buffer. Smears of each sediment were examined by immunofluorescence microscopy using a fluorescein-labelled rabbit antibody for a globulin fraction of fresh guinea-pig serum containing first component of complement.A greater degree of aggregation of the antigen-antibody complexes was observed in the sediments from tests with modified complement, that is complement supplemented with a diluted bovine serum globulin fraction prepared by dialysis. Aggregates from mixtures showing increased fixation of complement, as determined by titration of the residual hemolytic activity of the supernatant, appeared somewhat more brightly fluorescent. Very faint or no fluorescence was evident in the stained washed sediments from mixtures of antigen and antibody without complement or from mixtures of antigen, heat-inactivated normal bovine serum and complement.