Experiments correlating the quantity of serum nitrogen adding to specific precipitates of SIII with a measurement of the residual activity of each of the complement components after combination showed the following:

  1. C′1, C′2 and C′4 from whole serum contributed combining nitrogen to specific precipitates of SIII; C′1 from mid-piece also contributed combining nitrogen. C′2 and C′4, in the absence of C′1, did not contribute combining nitrogen; and C′3 contributed little or no combining nitrogen in any case.

  2. The decrease in the combining nitrogen per ml of serum which occurred as the volume of serum was increased, was primarily due to a diminution in the amount of C′1 fixed.

  3. Inactivation of C′1 of the CO2-insoluble fraction of serum by heating at 56 C for 60 minutes did not destroy the total combining capacity of the fraction. Sixty per cent of the combining nitrogen contributed by untreated mid-piece was still available after heat inactivation. Similar results were obtained when 1 ml amounts of heat-inactivated whole serum were fixed.

  4. The estimation of fresh serum nitrogen adding to specific precipitates is a measure of a variable portion of the total complement complex or of the combining components of complement. Therefore, it is necessary to determine the amount of residual activity of each component after fixation in order to interpret the data derived from combining complement nitrogen determinations.

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