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 conclusions about 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 human C5a ELISA kit is based on a catching monoclonal antibody that recognizes a neo-epitope on C5a-desArg. This prevents cross-reactivity with C5|
Cross reactant |
Reactivity |
|
Mouse, rabbit, rat and pig C5a |
Weak |
|
Horse C5a |
No |
|
Human C3a |
No |
- Working time of 3½ hours.
- Minimum concentration which can be measured is 0.3 ng/ml.
- Measurable concentration range of 0.3 to 20 ng/ml.
- Working volume of 100 µl/well.
- The human 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 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 C5a.
- Biotinylated tracer antibody will bind to the captured human 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 human C5a standards (log).
- The human C5a concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
- Upon receipt, store individual components at 2 - 8°C. Do not freeze.
- Do not use components beyond the expiration date printed on the kit label.
- The standard, tracer and streptavidin-peroxidase are stable in lyophilized form until the expiration date indicated on the kit label, if stored at 2 - 8°C.
- The exact concentration of the standard is indicated on the label of the vial and the certificate of analysis.
- Once reconstituted, the standard is stable for 12 hours, if stored at 2 - 8°C. For longer stability, we recommend to store aliquots at -20°C. Stored at -20°C, the standard will be stable for 1 month.
- Once reconstituted, tracer and streptavidin-peroxidase are stable for 1 month if stored at 2 - 8°C.
- Upon receipt, foil pouch around the plate should be vacuum-sealed and unpunctured. Any irregularities to aforementioned conditions may influence plate performance in the assay.
- Return unused strips immediately to the foil pouch containing the desiccant pack and reseal along the entire edge of the zip-seal. Quality guaranteed until expiration date if stored at 2 - 8°C.
Normal human plasma containing baseline levels of human C5a, were spiked with human C5a, in concentrations of 10 and 2.5 ng/ml. Samples with and without human C5a, were incubated for 1 hour at 37°C. Samples were measured using the ELISA. Recovery values for human C5a, ranged between 100% and 113% .
Serum
Normal human serum containing baseline levels of human C5a, were spiked with human C5a, in concentrations of 10 and 2.5 ng/ml. Samples with and without human C5a, were incubated for 1 hour at 37°C. Samples were measured using the ELISA. Recovery values for human C5a, ranged between 89% and 102% .
Activated serum
Normal activated human serum containing baseline levels of human C5a, were spiked with human C5a, in concentrations of 10 and 2.5 ng/ml. Samples with and without human C5a, were incubated for 1 hour at 37°C. Samples were measured using the ELISA. Recovery values for human C5a, ranged between 99% and 108% .
- Hartmann H et al; Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures. J Immunol Meth 1993, 166: 35
- Stove S et al: Circulating complement proteins in patients with sepsis or systemic inflammatory response syndrome. Clin Diag Lab Immunol 1996, 3:175
- Krug N et al; Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma. Am J Respir Crit Care Med 2001, 164:1841
- Hartmann H et al; Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures. J Immunol Meth 1993, 166: 35
- Stove S et al: Circulating complement proteins in patients with sepsis or systemic inflammatory response syndrome. Clin Diag Lab Immunol 1996, 3:175
- Krug N et al; Complement factors C3a and C5a are increased in bronchoalveolar lavage fluid after segmental allergen provocation in subjects with asthma. Am J Respir Crit Care Med 2001, 164:1841
| Related products | Cat # | |
|---|---|---|
| MASP-2, Human, ELISA kit | HK326 | |
| MBL/MASP-2, Human, ELISA kit | HK327 | |
| MBL, Human, ELISA kit | HK323-02 | |
| TCC, Human, ELISA kit | HK328 | |
| Complement factor H, Human, ELISA kit | HK342-02 | |
| MASP-3, Human, ELISA kit | HK339 | |
| C1q, Human, ELISA kit | HK356 | |
| H-ficolin, Human, ELISA kit | HK340 | |
| Factor H, 402H/Y variant detection, Human, ELISA kit | HK353 | |
| L-ficolin, Human, ELISA kit | HK336 | |
| C3a, Human, ELISA kit | HK354 | |