C5a, Human, ELISA kit

Quantity:
2 x 96 det.
Catalog #:
HK349-02

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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 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
Specifications
Product type Assays
Quantity 2 x 96 det.
Standard range 0.3 to 20 ng/ml
Detection level 0.3 ng/ml
Working volume 100 µl/well
Species Human
Cross reactivity Horse - No, Mouse - Weak, Pig - Weak, Rabbit - Weak, Rat - Weak
Application The human C5a ELISA kit is to be used for the in vitro quantitative determination of human C5a/C5a-desArg in serum and plasma.
Principle 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.
Storage and stability Product should be stored at 4 °C. Under recommended storage conditions, product is stable for at least six months.
References 1. Morad H et al; Time-course analysis of C3a and C5a quantifies the coupling between the upper and terminal Complement pathways in vitro. J Immunol Meth 2015, 427: 13
2. Hartmann H et al; Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures. J Immunol Meth 1993, 166: 35
3. Stove S et al: Circulating complement proteins in patients with sepsis or systemic inflammatory response syndrome. Clin Diag Lab Immunol 1996, 3:175
4. 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
Disease Infectious diseases, Nephrology
Applications
Application assays: The human C5a ELISA kit is to be used for the in vitro quantitative determination of human C5a/C5a-desArg in serum and plasma.
Principle: 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.
References
Perfomance: The linearity of the assay was determined by serially diluting a sample containing 331 ng/ml human C5a. The diluted samples were measured in the assay. The line obtained a slope of 1.0907 and a correlation coefficient of 0.9988.
Recovery: Plasma
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%.
References
References: 1. Morad H et al; Time-course analysis of C3a and C5a quantifies the coupling between the upper and terminal Complement pathways in vitro. J Immunol Meth 2015, 427: 13
2. Hartmann H et al; Rapid quantification of C3a and C5a using a combination of chromatographic and immunoassay procedures. J Immunol Meth 1993, 166: 35
3. Stove S et al: Circulating complement proteins in patients with sepsis or systemic inflammatory response syndrome. Clin Diag Lab Immunol 1996, 3:175
4. 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
Assay Manual
pdf
(Size: 0.63 MB)
Safety Data Sheet
pdf
(Size: 0.14 MB)
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