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Catalog # HK701

C5a, Pig, ELISA kit

748,001.232,00

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The complement system is an important factor in innate immunity. During complement activation of complement protein 5 (C5), C5 is proteolytically cleaved and the anaphylatoxic peptide C5a is generated.
C5a is a small polypeptide consisting of 74 aminoacids (11kDa) and is derived from the N-terminus of the alpha chain. C5a itself is very short-lived and in serum is cleaved rapidly into the more stable, though biologically still active C5a-desArg (also called acylation stimulating protein, ASP). Therefore, measurement of C5a-desArg allows reliable evaluation of the level of complement activation in the test samples. For convenience, both forms will be referred to in the following text as C5a.
C5a acts as a potent anaphylatoxin causing smooth muscle contraction, vasodilation, increased vascular permeability, basophil and mast cell degranulation and lysosomal enzyme release. In addition, C5a is a potent chemotactic factor for eosinophils, basophils and monocytes.
C5a is involved in inflammatory reactions seen in gram-negative bacterial sepsis, trauma, ischemic heart disease, post-dialysis syndrome and a variety of autoimmune diseases. Elevation of C5a is associated with increased cardiovascular risk in patients with advanced atherosclerosis. Also, C5a is closely associated with the capillary leak syndrome in leukemic children after bone marrow transplantation. C5a is also a marker in urine for predicting the onset of acute graft rejection after kidney transplantation.
Activation products of the complement cascade contain neo-epitopes that are not present in the individual native components. The pig C5a ELISA kit is based on a catching monoclonal antibody highly specific for C5a-desArg and is not cross-reactive with C5.

Application
The Pig C5a ELISA kit is to be used for the in vitro quantitative determination of porcine C5a in serum and plasma samples.

Principle
The Pig C5a 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 Pig C5a. Biotinylated tracer antibody will bind to the captured Pig C5a. 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 Pig C5a standards (log). The Pig C5a concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.

Linearity
The linearity of the assay was determined by serially diluting four samples. The diluted samples were measured in the assay. The CV% obtained ranged between 3.8 and 12.3%

Precision and reproducibility
For the intra-assay variation, two operators tested 3 different samples in the ELISA kit. The specification for this test is that the CV% of the average amount of the two operators should be <10%.

Product type
Assays
Quantity
1 x 96 det., 2 x 96 det.
Standard range
Measurable concentration range of 0.3 to 20 ng/ml.
Detection level
Minimum concentration which can be measured is 0.3 ng/ml.
Working volume
100 µl/well
Species
4
Cross reactivity
Human – No, Mouse – No, Rat – No
Storage and stability
Product should be stored at 4 °C. Under recommended storage conditions, product is stable for at least six months.
Precautions
For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result with the use of or derivation of this product.
Disease
Infectious diseases, Nephrology
Assay Manual
HK701
372 kb
Safety Data Sheet

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