The complement system mediates a number of essential biological functions that participate in host defense against infection, initiation of the inflammatory reaction, processing and clearance of immune complexes and regulation of the immune response. There are three pathways of complement activation: the classical pathway is initiated by immune complexes, the lectin pathway by surface bound mannan binding lectin and the alternative pathway by all the surfaces that are not specifically protected against it. Each complement pathway generates a C3 convertase, a serine protease that cleaves the central complement protein C3, and generates the major cleavage fragment C3b. C3b is an opsonin and part of one of the main convertases that drives the complement cascade. In the presence of complement regulatory molecules C3b may be further degraded sequentially to iC3b, C3c, C3dg and C3d. The disadvantage of most complement biomarkers is their short half-life, making reliable sample collection and measurements difficult. Unlike other C3 fragments, C3c does not bind to other structures like pathogens, cell surface (receptors) and other plasma proteins. Therefore, C3c is a stable complement biomarker which will appear in the fluid phase only, without interference of other C3 based products. The measurement of C3c provides evidence of (uncontrolled) complement activation and can be used as an indicator of an inflammatory state. The complement is a key element of the innate immune system. Inappropriate activation is pathologic and leads for example to various autoimmune diseases (e.g. HUS). There are also indications that C3 is associated with the cardiovascular system and Parkinson’s disease, making C3c a reliable and useful biomarker.
The C3c ELISA, based on a antibody highly specific for a epitope exclusively on C3c, is a straightforward ELISA which can be used in diseases where complement activation is an element in an inflammatory response.
The human C3c ELISA kit is to be used for the in vitro quantitative determination of human C3c in serum and plasma.
The human C3c ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ hours. The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay. Samples and standards are incubated in microtiter wells coated with antibodies recognizing human C3c. Biotinylated tracer antibody will bind to the captured human C3c. Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody. Streptavidin-peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB). The enzyme reaction is stopped by the addition of oxalic acid. The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human C3c standards (log). The human C3c concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The linearity of the assay was determined by serially diluting a sample containing human C1c. The diluted samples were measured in the assay.
Recovery was determined by mixing citrate AF003 plasma and M/66427 in a ratio of 75%/25%, 50%/50%, 25%/75% and 0%/100%. C3c was measured with capture HM2318 3 µg/ml. The recovery of the samples is between the acceptable values of 80-120%.