After 1 h, free C1q is separated from the precipitated bound C1q by centrifugation. The percentage of radioactivity precipitated corresponds to the C1q-binding activity of the sample and indicates the level of CICs. This test is particularly sensitive for CICs containing IgM, therefore explaining the high level of positivity usually observed in rheumatoid arthritis. CICs containing IgG4, IgA, IgD, and IgE are not detected. The technique is reasonably reliable, but C1q needs to be radiolabeled, a procedure that can inactivate the delicate complement molecule if harshly executed.

Cell-Binding Assays

These assays try to quantify the binding of CICs to cells.

Raji Cell-Binding Assay

The basis of the Raji cell-binding assay is the binding of CICs to C3b and C3bi receptors on a continuous lymphoblastoid cell line originally derived from a patient with BurkittÕs lymphoma (Raji cells) [56]. Raji cells are characterized by a lack of surface immunoglobulins, few or lowafÞnity receptors for IgG Fc, and a large number of receptors for complement. CICs bound to cells are quantitated with a labeled anti-IgG antibody. This test is particularly sensitive for CICs containing IgG. The technique is reasonably reliable, but Raji cell lines cultured in different laboratories are quite different and express different levels of C3 receptors. Therefore, data from different sources cannot be compared directly; for the same reason, longitudinal studies in a speciÞc laboratory can be done only if the line is continuously subcloned.

3.2.1.6 Complement

The complement cascade consists of a group of serum proteins that participate in inßammation through the actions of increased vascular permeability, chemotaxis, opsonization, and cell lysis [57, 58]. Once the Þrst component is activated, each component is activated by its predecessor. C4 can be activated by the classical pathway, which can be initiated by antigenÐantibody reactions. C3 can be activated by the classic pathway and the alternative pathway, which can be acti-

vated by microorganisms. Because the levels of complement components in various body ßuids may be decreased during complement activation, their measurements can give a rough index of disease activity under conditions in which complement activation is prominent.

Any disease that gives rise to CICs may show a hypocomplementemia, provided the CICs contain IgG or IgM antibodies capable of activating complement. Serial serum complement levels may be depressed in rheumatoid arthritis with vasculitis; [54, 59, 60] they also may be depressed in SLE exacerbations, particularly when there is renal involvement [61, 62]. Conversely, as the disease activity declines, there may be a parallel return of the complement level toward normal.

There are primarily two types of clinical assays for complement components: (1) tests that detect and quantify the presence of complement components (immunochemical assays) and (2) tests that determine functional or total hemolytic activity of the complete complement cascade (complement hemolytic 50% or CH50).

Quantitation Tests

The basis of immunochemical assays is the reaction of complement proteins with speciÞc antibodies [63]. The most widely used immunochemical assay is radial immunodiffusion. In this technique, monospeciÞc antibody directed against a complement protein is incorporated into an agarose gel, holes are punched in the gel, and test samples or known standards are placed in the holes. As the antigenic complement protein diffuses into the gel and encounters its speciÞc antibody, a precipitin ring forms, which is proportional to the concentration of complement placed in the hole. A standard curve is constructed from the size of the rings produced by samples of known concentration, and the concentration of complement in the test sample is determined. A more rapid and slightly more sensitive technique is electroimmunodiffusion. In this technique, the sample is unidirectionally electrophoresed into an antibody-containing gel, yielding a set of ÒrocketsÓ whose height is proportional to concentration. QuantiÞcation tests for complement components include measurements of C3 and C4 complement proteins.