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  • 产品名称：HumanComplementC3EFSIAKit(BlueFluorescentProbe)

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HumanComplementC3EFSIAKit(BlueFluorescentProbe)
详情介绍：
Purpose The AssayLite Human Complement C3 EFSIA (Endpoint Fluorescent Sandwich Immunoassay) Kit employs a quantitative sandwich fluorescent probe technique that measures complement C3 in human plasma, serum, and cell culture supernatants in one hour. A polyclonal antibody specific for human complement C3 has been pre-coated onto a 96-well opaque microplate with removable strips. Complement C3 in standards and samples is sandwiched by the immobilized antibody and a polyclonal antibody specific for complement C3 conjugated to a fluorescent probe. All unbound material is then washed away before the endpoint fluorescence is measured.
Brand AssayLite™
Sample Type Cell Culture Cells, Plasma, Serum
Analytical Method Quantitative
Detection Method Fluorometric
Characteristics Human Complement C3 EFSIA Kit (Blue Fluorescent Probe)
Components Human Complement C3 Microplate: A 96-well black polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human complement C3.
Sealing Films: Each kit contains 3 precut, pressure sensitive aluminum sealing films that can be cut to fit the format of the individual assay.
Human Complement C3 Standard: Human complement C3 in a buffered protein base (7.5 ?g, lyophilized).
Red Fluorescent Human Complement C3 Probe: 6 vials, lyophilized.
EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 mL, 1 bottle).
Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 mL, 1 bottle).
Stabilizing Solution: A ready-to-use solution to stabilize the fluorescent component (8 mL, 1 bottle).
Material not included Fluorescent microplate reader (Molecular Device Gemini XPS, Biotek FLx 800 & Synergy H1 have been evaluated, other readers have not been evaluated for performance).
Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel).
Deionized or distilled reagent grade water.
An incubator set at 37°C.
Alternative Name Complement C3 (C3 ELISA Kit Abstract)
Background Complement component 3 plays a central role in all three complement activation pathways. The C3 precursor contains 1663 amino acids and has a molecular weight of about 180 kDa. Human C3 has 77% identity to mouse C3 at the amino acid level. C3 is cleaved by C3 convertase into two activated fragments C3a and C3b. The anaphylatoxin C3a is a vasoactive peptide and a mediator of local inflammatory processes. The C3b fragment in complex with a receptor can bind covalently to pathogen surfaces to promote phagocytosis. Acquired C3 deficiency is associated with severe recurrent meningococci and pneumococci infections. Plasma C3 and C3a levels are elevated in cryptogenic and large-vessel disease subtypes of ischemic stroke.
Gene ID 718
UniProt P01024
Research Area Complement Factors
Application Notes Normal human complement C3 plasma levels range from 0.5- 1.8 mg/mL.
Comment

Assay Type: Singleplex
Assay Time 1 h
Plate Pre-coated
Reagent Preparation

Freshly dilute all reagents and bring all reagents to room temperature before use.
EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8°C.
Standard Curve: Reconstitute the 7.5 µg of Human Complement C3 Standard with 2.5 mL of EIA Diluent to generate a working solution of 3 µg/mL. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (3 µg/mL) 1:2 with EIA Diluent to generate 1.5, 0.75, 0.375, 0.188, 0.094, and 0.047 µg/mL solutions. EIA Diluent serves as the zero standard (0 µg/mL). Any remaining solution should be frozen at -20°C and used within 30 days.
Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. Dilute samples 1:2000 into EIA Diluent and assay (suggested dilution factor only, the correct dilution factor should be determined by user). The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant).
Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes and remove serum. Dilute samples 1:2000 into EIA Diluent and assay (suggested dilution factor only, user should determine optimal dilution factor). The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Refer to Sample Dilution Guidelines below for further instruction.
Assay Procedure

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is incubated at 37°C.
Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator.
Add 100 µl of Human Complement C3 Standard or sample per well. Cover wells with a sealing film and incubate for 30 minutes at 37°C. Start the timer after the last addition.
Wash five times with 200 µl of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 µl of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid.
Add 100 µl of Fluorescent Human Complement C3 Probe per well. Cover wells with a sealing film and incubate for 30 minutes at 37°C. Start the timer after the last addition.
Wash the microplate as described above.
Immediately add 50 µl of Stabilizing Solution to each well.
Read the endpoint fluorescence on a microplate reader at an excitation wavelength of 488 nm and emission wavelength of 576 nm within 2 hours.
For the Gemini XPS reader, set PMT to High. For the FLx800 reader, a gain of 100 is suggested. For the Synergy H1, a gain of 140 is suggested. However, the user should determine the optional gain/ sensitivity.
Calculation of Results

Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 576 emitted fluorescence on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor.
Restrictions For Research Use only
Precaution of Use Prepare all reagents (working diluent buffer, wash buffer, standards, and fluorescent probe) as instructed, prior to running the assay.
Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor.
This kit is for research use only.
The kit should not be used beyond the expiration date.
Avoid direct light exposure to the assay.
Store the Fluorescent Probe in a dark place. Do not freeze.
Storage 4 °C
Storage Comment Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date.
Store Microplate, Diluent Concentrate (10x), Stabilizing Solution, and Wash Buffer at 2-8°C.
Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator.
Diluent (1x) may be stored for up to 30 days at 2-8°C.
Store the standard and at 2-8°C before reconstituting with diluent and at -20°C after reconstituting with diluent.
Store the fluorescent probe in a dark place at 2-8°C before and after reconstituting with diluent. Do not freeze.
Supplier Images
ELISA image for Human Complement C3 EFSIA Kit (Blue Fluorescent Probe) (ABIN1774746) Human Complement C3 EFSIA Kit (Blue Fluorescent Probe)
ELISA image for Human Complement C3 EFSIA Kit (Blue Fluorescent Probe) (ABIN1774746) Human Complement C3 EFSIA Kit (Blue Fluorescent Probe) (Image 2)