The complement system plays important roles in both innate and adaptive immune response and can produce an inflammatory and protective reaction to challenges from pathogens before an adaptive response can occur. It consists of a complex family of proteins and receptors which are found in the circulation, in tissues and other body-fluids. There are three pathways of complement activation. The classical pathway is initiated by Immune complexes; the lectin pathway by surface bound lectins; and the alternative pathway by all the surfaces that are not specifically protected against it. Each generates a C3 convertase, a serine protease that cleaves the central complement protein C3, and generates the major cleavage fragment C3b. The C3 and C5 convertases are enzymatic complexes that initiate and amplify the activity of the complement pathways and ultimately generate the cytolytic MAC.
The synthesis of C3 is tissue-specific and is modulated in response to a variety of stimulatory agents. After cleavage by C3 convertase the anaphylotoxin C3a and activating C3b are formed. When bound to the cell surface C3b forms the start of the terminal pathway of complement by initiating the formation of the C5 convertase. Further cleavage of C3b by trypsinlike enzymes lead to formation of iC3b and subsequently C3c and C3dg. The latter digested to leave C3d. The formation of C3dg into C3d in blood is a slow step. As a result the majority will be C3dg.
C3 has a molecular weight of app. 185kDa and is the most abundant protein of the complement system with serum protein levels of about 1.3 mg/ml. C3dg is a non-glycosylated single chain protein of 38.9 KDa. Most assays cannot distinguish between the different C3 proteins. C3 activation products are involved in a number of diseases like transplantation rejection, kidney diseases, AMD and inflammatory diseases. Surface bound C3 proteins also have role, eg via complement receptor2 (CR2), in regulating the adaptive immune response. Therefore complement fragments serve as biomarkers for many of these diseases. The binding of C3dg to the cell membrane is rather unstable, leading to release of the protein. As end product of C3 activation, this makes it an attractive diagnostic biomarker. Instead of C3 & C4 it reflects more ongoing complement activation. Most antibodies, although recognizing an epitope on the C3d part of the alpha chain, do not differentiate between the native and activated C3 proteins.
The Human C3d ELISA kit is to be used for the in vitro quantitative determination of C3d in plasma and serum samples.
The Human C3d ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 1 hour and 15 minutes.
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 C3d.
Peroxidase-conjugated antibody will bind to the captured C3d.
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 C3d standards (log).
The Human C3d concentration of samples, which are run concurrently with the standards, can be
determined from the standard curve.