Complement is a set of serum proteins that undergo a sequential series of cleavage and association reactions in response to antibody-antigen complexes. If these complexes occur on a cell surface, complement reactions can cause cell lysis (1). The lysis of erythrocytes is particularly easy to detect visually, due to the release of hemoglobin, and this lysis forms the principle for a type of immunoassay. Antibody-antigen complexes in solution cause complement reactions to occur without cell lysis. Since the complement reactions are stoichiometric and the products short-lived, these non-cell-associated complexes deplete a portion of the complement proteins, a process termed "complement fixation." This depletion reduces the lysis observed when the complement is subsequently added to antibody-coated erythrocytes.
Key reagents for complement fixation assays, all available commercially, are complement, erythrocytes, and hemolytic antibody (2, 3). Guinea pig serum is usually used as a source of complement, since guinea pig complement reacts with antibodies of most experimentally significant mammalian species. Sheep erythrocytes, washed free of serum components, are used for lysis. Hemolytic antibody is obtained by immunizing rabbits with sheep erythrocyte membranes. The erythrocytes are sensitized in preparation for complement lysis by incubation with the hemolytic antibody. Once amounts of complement and sensitized erythrocytes appropriate for easy detection of lysis are determined, the reagents can be used to measure the interaction of an antibody-antigen pair (4). The antibody and antigen to be tested are incubated for a set time in the presence of complement. Standards containing known amounts of antibody and antigen are incubated simultaneously. Sensitized erythrocytes are next added, and lysis is measured after further incubation, by spectrophotometric determination of hemoglobin. Diminished lysis relative to a control indicates that the antibody-antigen pair in question has formed an aggregate and induced complement reactions to occur in solution, reducing the amount of complement available to lyse cells. Results observed with the standards are used to calibrate the correspondence between antibody-antigen complex formation and lysis. Because antibody-antigen aggregates are the most active species in fixing complement, bell-shaped response curves result when percent complement fixed is plotted against added antibody or antigen. Complement fixation is most efficient at roughly equivalent amounts of antibody and antigen, whereas either component in excess will favor formation of binary and ternary complexes that do not fix complement well. Consequently, several concentrations of antigen or antibody must be tested to establish whether the observed response lies on the increasing or decreasing slope of the standard curve.
As an analytical technique, complement fixation is sensitive to nanogram quantities of antigen, but is subject to reagent variation and interference by chemical components of the sample. A notable value, however, is in study of cross-reactivity between structurally similar antigens. Proteins that differ slightly in sequence give easily measured differences in complement fixation ability when they are probed with a single antiserum. This effect is so regular that complement fixation by homologous antigens can be used to establish the evolutionary relationship between closely related species (5, 6).
