Name
C3a, Human, ELISA kit
Catalog nr
HK354
(lot number and expiry date are indicated on the label)
Short description
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 p...
Size
2 x 96 det
Application
Manual
Description
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.
Cross Reactivity
No cross reactivity with C5a and C3.
Formulation
2 x 96 det
Application
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. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.
Features
Working time of 3½ hours.
Minimum concentration which can be measured is 31.3 pg/ml.
Measurable concentration range of 31.3 to 2000 pg/ml.
Working volume of 100 µl/well
Minimum concentration which can be measured is 31.3 pg/ml.
Measurable concentration range of 31.3 to 2000 pg/ml.
Working volume of 100 µl/well
Typical standard curve
Principle
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.
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.
Storage and stability
- 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 quality control.
- 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.
References
- 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 at al; Re-evaluation of the storage conditions for blood samples which are used for determination of complement activation. J Immunol Meth 1995, 182: 1
Also available
References
- 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 at al; Re-evaluation of the storage conditions for blood samples which are used for determination of complement activation. J Immunol Meth 1995, 182: 1
Scientific info
A reliable measurement of complement activation
Anaphylatoxin C3a
- C3a is a potent anaphylatoxin of 74 amino acids
- C3a is rapidly cleaved int
- o the less potent but more stable C3a-desArg
- Central activation product of all three different complement pathways
- Normal C3a values in plasma range between 48 and 150 ng/ml (median 86.4 ng/ml ± 29.3)
- C3a is a mediator of local inflammatory processes
- C3a binds to specific cell surface receptors and causes leukocyte activation, histamine release, smooth muscle contraction and vascular permeability
C3a and diseases
- Elevated levels of C3a are correlated to higher risk of developing AMD
- During exacerbations of systemic lupus erythematosus (SLE) levels of C3a are increased
- Involved in the pathogenesis of asthma
- Increased levels of C3a are correlated to sepsis severity
- C3a has been implicated in myocardial ischemia injury, rheumatoid arthritis, inflammatory bowel disease and psoriasis
- C3a was found to be elevated in patients with chronic hepatitis C and HCV-related hepatocellular carcinoma
Matrix influences in human C3a ELISA
- Different matrices were evaluated in the human C3a ELISA
- Serum and plasma have to be diluted 4000x and 300x respectively to obtain reliable measurement
- C3a in bronchoalveolar lavages (BAL) can be detected accurately when BAL is diluted at least 5x
- C3a in urine can be detected accurately when urine is diluted at least 4 times
- No influence of anticoagulant was detected
- Recovery of spiked human C3a in different matrices was within the acceptable range of 80-120 % (Table 1)
Table 1 Recovery of spiked human C3a in different matrices
Reference standard
- Purified C3a derived from Complement Technology Inc was used to validate our standard calculation (Fig 1)
- Perfect match between external C3a source and Hycult Biotech C3a standard (R2= 0.9996)
Hallmarks of human C3a ELISA (Cat.#HK354)
- Working time of 3.5 hours
- 100 µl sample/well
- Detection limit of 31.3 pg/ml
- Measurable range of 31.3-2000 pg/ml
- Useful for serum, plasma, urine and BAL
- Detection of C3a as well as C3a-desArg
- No interference by C3 due to neo-epitope detection
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