C5a is a protein fragment released from complement component C5. This gene is mapped to 9q33.2. The protein encoded by this gene is the fifth component of complement, which plays an important role in inflammatory and cell killing processes. This protein is comprised of alpha and beta polypeptide chains that are linked by a disulfide bridge. An activation peptide, C5a, which is an anaphylatoxin that possesses potent spasmogenic and chemotactic activity, is derived from the alpha polypeptide via cleavage with a convertase. The C5b macromolecular cleavage product can form a complex with the C6 complement component, and this complex is the basis for formation of the membrane attack complex, which includes additional complement components. Mutations in this gene cause complement component 5 deficiency, a disease where patients show a propensity for severe recurrent infections. Defects in this gene have also been linked to susceptibility to liver fibrosis and to rheumatoid arthritis.
Principle of the Assay:The Complement C5a (C5a), BioAssay™ ELISA Kit (Human) is based on standard sandwich enzyme-linked immunosorbent assay technology. A mouse monoclonal antibody specific for C5a has been pre-coated onto 96-well plates. Standards (E. coli-expressed recombinant fragment corresponding to T678 thru R751 of human C5a) and test samples are added to the wells, incubated and then biotinylated detection goat polyclonal antibody specific for C5a is added, followed by an incubation step and a washing step with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. HRP substrate TMB is used to visualize the HRP-mediated reaction. TMB is catalyzed by HRP to produce a blue colored product that changes into yellow upon addition of acidic stop solution. The intensity of the yellow color is proportional to the amount of human C5a in the sample.
This kit is intended for the quantitative determination of human C5a in cell culture supernatants, cell lysates, tissue homogenates, serum and plasma (heparin, EDTA).
Storage and Stability:Unopened kit is stable at 4°C for 6 months.
Assay Summary:1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash. 2. Add biotinylated antibody and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS or PBS
3. Add ABC working solution and incubate the plate at 37°C for 30min. Wash plate 5 times with 0.01M TBS or PBS
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 20-25 min
5. Add Stop solution and read absorbance at 450nm.
