Complement C5 (molecular weight app. 190kDa) is a key component of the complement system. This 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. The complement system consist of a complex family of proteins and receptors which are found in the circulation, in tissues and other body-fluids. Excessive and uncontrolled activation/regulation of the complement system have been implicated in various diseases such as asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome and ischemia-reperfusion injuries, and rejection of transplanted organs. Three major pathways within the complement system have been described, namely the classical, alternative and lectin pathways which are activated by different stimuli. The classical pathway is initiated by Immune complexes; the lectin pathway by surface bound lectins; and the AP by all the surfaces that are not specifically protected against. All the 3 pathways converge on Complement C3. Cleavage of C3 induces, in turn, the cleavage of C5, successively leading to the production of the membrane attack complex (MAC). This protein complex is the final common effector responsible for lysis of susceptible microorganisms or damage cells. Mutations in C5 gene cause complement component 5 deficiency, a disease characterized by recurrent bacterial infections.
The Human C5 ELISA kit is to be used for the in vitro quantitative determination of C5 in plasma and serum samples.
The Human C5 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 C5.
Biotinylated tracer antibody will bind to the captured Human C5.
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 C5 standards (log).
The Human C5 concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Normal human blood samples (plasma) containing baseline levels of human C5, were spiked with human C5, in concentrations of 100 and 12.5 ng/ml. Samples with and without human C5, were incubated for 30 minutes at room temperature. Samples were measured using the ELISA. Values for human C5, ranged between 96% and 109%.