Complement C4D ELISA Kit [Human] (CAT#: CB-P057-K) Datasheet

Product Type
Kit
Description
The complement system plays an essential role in autoimmune and infectious diseases. There are three pathways of complement activation; the classical, the alternative and the lectin pathway. The C4d protein is a product of the classical and the lectin pathways. C4d, a degradation end product produced during complement C4 activation, had been recognized as a biomarker for its stability and strong association with antibody-mediated rejection in the 1990s and in the last twenty years, the potential importance of C4d in SLE as a tool for diagnosing and monitoring SLE was highlighted. Particularly, C4d associates with SLE nephritis. In primary Sjogren's syndrome (pSS) the level of C4d correlates with anti-SSB and κ/λ ratio and is suggested to be an appropriate marker of antibody response and complement activation in pSS patients with auto-antibodies. Plasma level of C4d has been shown to be significantly higher in patients with antibody-associated vasculitis with active disease compared with patients with lupus nephritis and normal controls. C4d has the potential to detect patients at risk for the consequences of antibody-mediated disease. Furthermore, the development of new therapeutics that block complement activation makes C4d a marker with potential to identify and monitor patients who may possibly benefit from these drugs. Complement assays based on detection of linear neoepitopes has been reported to have an advantage compared to conformational epitopes, as it reduces the risk of false positives and increases specificity. This complement assay is based on the detection of the short linear C4d neoepitope exposed at the cleavage site of C4 after activation. The Complement C4d assay is an enzyme-linked immunosorbent assay (ELISA) for the semiquantitative determination of C4d in human plasma.
Applications
This kit is for research use only and cannot be used for diagnosis.
This kit has been used to test EDTA plasma. Other matrices have not been tested.
Principle
The device 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 C4d in the sample is captured by the anti-C4d-Neo 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, that binds to both
allelic variants of C4d (A and B), is added the 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 C4d bound to the well. The amount of C4d is determined by comparison to the color
development of the calibrator samples.
Detection Method
Enzyme immunoassay (ELISA) technique
Detection Limit
0 / 5 - 400 ng/mL
Sample
Plasma
Sample Volume
10 µL
Controls
1 low control (ready to use) and 1 high control, ready to use.
Species Reactivity
Human
Cross Reactivity
Analytical specificity of the Complement C4d assay was challenged by analyzing complement factors genetically related to C4d. The following factors were analyzed: C4, C4b, C3, C3b, C3d, C5, SC5b-9 and C9. To challenge cross-reactivity the complement factors were tested at concentrations just above physiological concentrations. Note that the Complement C4d assay detects both genetic variants of C4d (A and B).
Protocol Length
1 hour incubation (37°C) + 30 min. (RT) + 30 min. (RT) = 2 hours total incubation time.
Components
- One frame with micro titer wells (12x8) coated with anti-C4d-Neo monoclonal antibody, sealed in a foil pack with a dessiccation bag.
- 1,5 mL Low control (LC). Ready to use
- 1,5 mL High control (HC). Ready to use
- 2x30 mL Diluent (Dil), red colour. Ready to use.
- 15 mL Conjugate containing HRP-labelled antibodies to C4d. 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 1,5 mL vials with calibrators containing human recombinant C4d,
- 0 ng/mL, 5 ng/mL, 25 ng/mL, 100 ng/mL, 200 ng/mL and 400 ng/mL. Ready to use
Requires
-Materials or equipment required but not provided
- Microplate reader with filter 450nm and 620nm
- 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. When stored at 2-8°C the diluted wash
solution is stable until the date of expiration of the kit.
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.

Table 1. Reference range of Complement C4d assay


Table 2. Within-run and between-run precision. Note: Results below are mean concentrations at 1/100 dilutions according to assay procedure.


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


Table 4. Linearity and recovery. Four C4d containing samples were diluted in diluent. Theoretical concentrations were calculated based on the 1/100 dilution.

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

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