gC1qR is present in plasma and the extracellular matrix. The molecule is an unusually acidic, single chain protein with an apparent molecular weight of 33 kDa. It does not possess a conventional sequence motif compatible with a membrane spanning segment nor a consensus site for a GPI anchor. gC1qR migrates as a single chain under both reducing and non-reducing conditions, but it behaves as an oligomer on gel-filtration in non-dissociating conditions.
Its multimer formation may be a critical process by which the gC1qR molecule increases its affinity for multivalent ligands such as C1q. gC1qR has been shown to inhibit complement activation by preventing the binding of C1q to antibodies, suggesting that the binding site for gC1qR and the binding site for immune complexes, which are present on the C1q globular ‘heads’, may be located at the same position. gC1qR is capable of interacting with several proteins involved in blood clotting, namely, thrombin, prothrombin, the heparin binding form of vitronectin, the ternary complex, vitronectin-thrombin-antithrombin, as well as high-molecular-weight kininogen and Hageman factor.
Besides its role in the complement pathway, gC1qR participates in enhancement of Fc-receptor and CR1-mediated phagocytosis, procoagulant activity on platelets, and chemotactic activity on mast cells, eosinophils, neutrophils, and fibroblasts. gC1qR is expressed on a wide variety of cells and can serve as a binding site for plasma and microbial proteins. Its antigenic sites may be cryptic on cells in suspension but become exposed when the cells are activated or fixed by bifunctional cross-linkers.
Probably it is also expressed on the cell membrane as a trimer. Crosslinking or activation may thus bring about a trimeric assembly of gC1qR followed by a conformational change within the molecule, thereby exposing epitopes which are otherwise hidden. A form of gC1qR is also found inside the cell. Intracellular gC1qR has been shown to bind the cytoplasmic tail of the α1B-adrenergic receptor and to PKCμ.
The human gC1qR ELISA kit is to be used for the in vitro quantitative determination of human gC1qR in plasma samples and cell culture supernatant.
The human gC1qR 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 gC1qR. Biotinylated tracer antibody will bind to the captured human gC1qR. 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 gC1qR standards (log). The human gC1qR concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The recovery of human gC1qR from an undiluted sample is not 100% and may vary from sample to sample. When testing less diluted samples it is advisable to run recovery experiments to determine the influence of the matrix on the detection of human gC1qR.
Automated ELISA performance
In case plate washer is used, please note: use of a plate washer can result in higher background and decrease in sensitivity. We advise validation of the plate washer with the manual procedure. Make sure the plate washer is used as specified for the manual method.