Serum complement has traditionally been defined as a four component system, Cq, C'2, C~3, and C~4. Although several investigators had suggested the existence of additional components (for review of earlier literature, see reference 1), only recently has unequivocal evidence been available supporting the further complexity of complement. The third component, C'3, was shown first to consist of at least two activities (1--4) and then three (5, 6), designated C~3a, C'3b, and C'3c. A highly purified protein,/31o-globulin (7), would appear to be identical with C'3a (5).Miiller-Eberhard and Kunkel (8, 9) and Taranta, Weiss, and Franklin (10) have described a new serum protein, distinct from the classical components of complement and designated the llS component on the basis of its sedimentation constant. In earlier work on the sequence of action of the components of complement (11, 12), the first component, C'I, was the first factor recognized to interact with sensitized erythrocytes. However, evidence has been presented that the I1S component participates in immune hemolysis at a stage even earlier than Ctl (8, 9), an observation which would appear to require revision of previous concepts of sequence.It is the purpose of this paper to report the resolution of Cq into three activities by chromatography on diethylaminoethyl (DEAE) cellulose of a euglobulin fraction of normal human serum. It will be shown that all three factors are required for reconstitution of Cq activity in several hemolytic systems and for generation of Cq esterase in the absence of antigen-antibody complexes (13).

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Serum complement is of potential importance to clinical medicine and to human biology in immune reactions and hypersensitivity states. However, most studies on the characteristics and mechanism of action of complement have been performed on systems largely of non-human origin (1, 2). The present studies were undertaken to study a specific immune human hemolytic disorder and to use this as a model system for the study of human complement in a reaction system involving human erythrocytes and an antibody of human origin. The Donath-Landsteiner (D-L) reaction (3), responsible for the clinical syndrome of paroxysmal cold hemoglobinuria, is a reaction which requires for hemolysis that erythrocytes be incubated with antibody and fresh serum complement first at low temperatures, usually near 0°C., and then at higher temperatures, usually 37°C. It was chosen as the model for these studies because: (a) it involves human ceils and antibody and avoids the use of reagents from multiple species (rabbit antibody, sheep red cells, guinea pig complement); (b) the D-L antibody is an active hemolysin but a weak agglutinin in contrast to the isoantibodies and cold agglutinins; and (c) the biphasic nature of the reaction permits the separation of the early and late phases of complement action.The studies in the present paper were undertaken to define the kinetics of the Donath-Landsteiner reaction and served as the basis for the observations on the mechanism of action of complement in the succeeding paper. The data indicate that many conflicting earlier observations regarding the role of complement in the reaction (3--8) may be explained on the basis of variation in (a) potency and amount of antibody used, (b) sensitivity of cells, (c) amount

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Hypersensitivity responses to two highly purified and well characterized mycobacterial proteins and a preparation of mycobacterial arabinogalactanarabinomannan were studied by using guinea pig skin tests and in vitro-cultured human lymphocyte mitogenesis stimulation assays. The purified proteins were found to elicit delayed-type hypersensitivity skin test reactions and to stimulate mitogenesis in cultured lymphocytes. The polysaccharide preparation was found to evoke delayed skin tests but not to stimulate cultured lymphocytes.