The complement system is an important factor in innate immunity. The third complement component, C3, is central to the classical, alternative and lectin pathways of complement activation. The synthesis of C3 is tissue-specific and is modulated in response to a variety of stimulatory agents. During complement activation, C3 is proteolytically cleaved resulting in release of the anaphylatoxic peptide C3a.
C3a is a small polypeptide consisting of 74 amino acids. C3a itself is very short-lived and in serum cleaved rapidly into the more stable C3a-desArg (also called acylation stimulating protein, ASP) . Therefore, measurement of C3a-desArg allows reliable conclusions about the level of complement activation in the samples. For convenience, both forms will be referred to in the following text as C3a.
C3a is a mediator of local inflammatory processes. It induces smooth muscle contraction, increases vascular permeability, and causes histamine release from mast cells and basophilic leukocytes. C3a is involved in inflammatory reactions seen in gram-negative bacterial sepsis, trauma, ischemic heart disease, post-dialysis syndrome and a variety of autoimmune diseases. Normal values in plasma of control persons range between 48 – 150 ng/ml (median: 86.4 ng/ml, SD: 29.3 ng/ml).
Activation products of the complement cascade contain neo-epitopes that are not present in the individual native components. The human C3a ELISA kit is based on a catching monoclonal antibody that recognizes a neo-epitope on C3a-desArg. This prevents cross-reactivity with C3.
The human C3a ELISA kit is to be used for the in vitro quantitative determination of human C3a/C3a-desArg in serum, plasma, bronchoalveolar lavage fluid (BALF) and urine samples.
The human C3a 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 2 plates 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 C3a.
Biotinylated tracer antibody will bind to captured human C3a.
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 C3a standards (log).
The human C3a concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.