Complement TCC/sC5b-9 [Human] (CAT#: CB-P062-K) Datasheet

Product Type
Kit
Description
In all cases where determination of complement function is wanted, TCC levels can be very informative as a supplement to functional assessment of the three complement pathways. It reflects the historical in vivo activity of complement in a given sample. The complement system plays an essential role in chronic, autoimmune and infectious disease. There are three pathways of complement activation, namely the classical, the alternative and the lectin pathway. The soluble terminal complement complex is a product of the terminal pathway and can be a result from all three complement activation pathways. Since TCC reflects activation to the end of the final terminal pathway irrespective of the initial pathway involved, it is a particularly good candidate for general evaluation of complement activation. It is well-known that the complement system plays a key role in the development and amplification of the inflammatory process at the tissue level in various pathological conditions. Increased levels of TCC can be detected in for example hemolytic uremic syndrome (HUS), Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). The complement system can also be activated by artificial surfaces, for example during hemodialysis or cardiopulmonary bypass, resulting in increased levels of TCC. TCC is also well suited for studies of complement activation by biomaterials in medical devices. TCC can like other activation products of complement be measured in assays using neo-epitope specific monoclonal antibodies. Neoepitopes are hidden in the native complement component and exposed after complement activation.
Applications
Complement TCC is an enzyme immunoassay for the semi-quantitative determination of soluble terminal complement complex (sTCC, also known as TCC or sC5b-9) in human EDTA plasma. For research use only, not for diagnostic procedures.
Principle
The assay is a colorimetric sandwich ELISA kit. Samples are diluted in assay diluent and 100µL diluted sample is transferred to the microtiter wells and incubated at room temperature for 60 minutes. During this first incubation TCC in the sample is captured by the anti-TCC monoclonal antibody, pre-coated on the surface of the microtiter wells. After washing to remove unbound material, a second horseradish peroxidase (HRP) labelled monoclonal antibody is added to detect the TCC bound to the well. After incubation for 30 minutes the wells are washed again and a substrate is added and incubated. The color development is stopped after 30 minutes and the color is measured in a spectrophotometer. The color is directly proportional to the amount of TCC bound to the well. The amount of TCC is determined by comparison to the color development of the calibrator samples.
Detection Method
Enzyme immunoassay (ELISA) technique
Detection Limit
0 / 10 - 400 ng/mL
Sample
Plasma
Sample Volume
30 µL
Controls
1 low control (ready to use) and 1 high control, ready to use.
Species Reactivity
Human
Protocol Length
1 hour incubation (37°C) + 30 min. (RT) + 30 min. (RT) = 2 hours total incubation time.
Components
-One frame with microtiter wells (12x8) coated with anti-TCC monoclonal antibody, sealed in a foil pack with a dry pack.
- 1.5mL Low control (LC). Ready to use.
- 1.5mL High control (HC). Ready to use.
- 2 x 30 mL Diluent (Dil). Ready to use.
- 15 mL Conjugate containing HRP-labelled antibodies to TCC. Ready to use.
- 15 mL Substrate TMB. Ready to use.
- 15 mL Stop solution (0.5M H2SO4). Ready to use.
- 30 mL Wash solution, 30x concentrated.
- 6 x 1.5mL vials with calibrators containing human purified TCC, 0 ng/mL, 10ng/mL, 50 ng/mL, 100ng/mL, 200ng/mL and 400ng/mL. Ready to use.
Requires
- Microplate reader with filter 405 nm.
- Precision pipettes with disposable tips.
- Washer for strips, absorbent tissue, tubes and a timer.
Handling Advice
All reagents in the kit are ready to use except the wash solution. The reagents should be stored at 2-
8°C. Components from different lots shall not be mixed.
Storage
2 - 8 °C
Research Use
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.

Figure 1. Example of a calibrator curve

Table 1. Plasma from 144 blood donors were tested and the normal 95% reference range was calculated

Table 2. Between-run and within-run precision. Note: results below are mean concentrations at 1/100 dilution according to assay procedure.

Table 3. Batch to batch precision. Note: results below are mean concentrations at 1/100 dilution according to assay procedure.

Table 4. Linearity and dilution recovery.
A dilution series was prepared for three EDTA plasma samples. A 1/10 dilution has been used in all reported results in the IFU but dilutions between 1/10 and 1/40 will yield accurate TCC concentrations (Table 4). Note that using a different sample dilution than 1/10 may shift the reference range. It is recommended that each laboratory establish a reference range with the dilution of choice.

Table 5 Non-interfering substances
The substances in Table 5 were tested in the Complement TCC RUO assay and not found to interfere.

All products and services are for Research Use Only. Do Not use in humans.

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