The mouse C3b ELISA assay is specific for the cleaved C3 fragments C3b, iC3b, and C3c, and for activated C3.
The complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. C3b is further cleaved into iC3b, C3c, C3dg and C3f. C3b and iC3b function as opsonins, they act through different complement receptors, complement receptor 1 (CD35) and complement receptor 3 (CD11b/CD18), respectively.
The mouse C3b ELISA assay is specific for the cleaved C3 fragments C3b, iC3b, and C3c, and for activated C3. Therefore, positive reactivity in plasma or serum is associated with activation of the complement cascade and C3 cleavage. In case of an acute inflammatory reaction, lots of C3 are processed into the products recognized by the assay, making this assay a useful tool for measuring the acute inflammatory response. The assay is less useful for assessing chronic inflammatory conditions since minimal reactivity may be observed. In such cases, primarily the C3dg product resides at the place of inflammation (C3c being cleared) which is not recognized by the assay. The chronic processing/activation of C3 is taking place at a lower level, which would reduce detection of the C3 fragments C3b, iC3b, and C3c. Beware that complement activity levels are mouse strain dependent and might be affected by the way the samples are collected and processed. C3 levels can differ between healthy and diseased animals, the optimal dilution can be different between these statuses.
The Mouse C3b ELISA kit is to be used for the in vitro quantitative determination of C3b in serum and plasma samples.
The Mouse C3b 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 Mouse C3b. Biotinylated tracer antibody will bind to the captured Mouse C3b. 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 Mouse C3b standards (log). The Mouse C3b 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 samples containing mouse C3b. The diluted sample was measured in the assay. Samples were diluted at least 450 times. The CV% ranged between 0.6 and 9.8%.
Normal mouse serum samples containing baseline levels of mouse C3b, were spiked with mouse C3b, in concentrations of 31 and 250 AU/ml. Samples with and without mouse C3b were incubated for 30 minutes at room temperature and measured using the ELISA. Values for mouse C3b, ranged between 96% and 115%.