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C3 ELISA Kit

C3 Reactivity: Human Colorimetric Competition ELISA Cell Culture Supernatant, Plasma
Catalog No. ABIN612675
  • Target See all C3 ELISA Kits
    C3 (Complement Component 3 (C3))
    Reactivity
    • 6
    • 5
    • 5
    • 4
    • 4
    • 3
    • 3
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    Human
    Detection Method
    Colorimetric
    Method Type
    Competition ELISA
    Minimum Detection Limit
    0.5 µg/mL
    Application
    ELISA
    Purpose
    The AssayMax Human Complement C3 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human complement C3 in plasma, serum and cell culture supernatants
    Brand
    AssayMax
    Sample Type
    Plasma, Cell Culture Supernatant
    Analytical Method
    Quantitative
    Cross-Reactivity (Details)
    No significant cross-reactivity or interference was observed.
    Components
    Human Complement C3 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human complement C3. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Complement C3 Standard: Human Complement C3 in a buffered protein base (30 µg, lyophilized). Biotinylated Complement C3: 1 vial, lyophilized. EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). 1 Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
    Material not included
    Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water
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  • Sample Volume
    25 μL
    Assay Time
    < 3 h
    Plate
    Pre-coated
    Protocol
    This assay employs a quantitative competitive enzyme immunoassay technique that measures human complement C3 in less than 3 hours. A polyclonal antibody specific for human complement C3 has been pre-coated onto a 96-well microplate with removable strips. Complement C3 in standards and samples is competed by a biotinylated complement C3 sandwiched by the immobilized antibody and streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
    Reagent Preparation

    Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 30 g of Complement C3 Standard with 1 ml of EIA Diluent to generate a solution of 30 g/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 (30 g/ml) 1:2 with EIA Diluent to produce 15, 7.5, 3.75, 1.88, 0.94 and 0.47 g/ml solutions. EIA Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Complement C3] ( g/ml) Standard (30 g/ml) P1 30.000 P2 1 part P1 + 1 part EIA Diluent 15.000 P3 1 part P2 + 1 part EIA Diluent 7.50 P4 1 part P3 + 1 part EIA Diluent 3.75 P5 1 part P4 + 1 part EIA Diluent 1.88 P6 1 part P5 + 1 part EIA Diluent 0.94 P7 1 part P6 + 1 part EIA Diluent 0.47 P8 EIA Diluent 0.00 Biotinylated Complement C3 (2x): Dilute Biotinylated Complement C3 with 4 ml EIA Diluent to produce a 2-fold stock solution. Allow the biotin to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution should be further diluted 1:2 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.

    Sample Collection
    Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:800 into EIA Diluent. 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 anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:800 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
    Assay Procedure

    Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 25 µL of standard or sample per well, and immediately add 25 µL of Biotinylated Complement C3 to each well (on top of the Standard or sample) and mix gently. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 8 minutes or until the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

    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 450 nm absorbance 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

    Assay Precision
    Intra-assay and inter-assay coefficients of variation were 4.5 % and 7.4 % respectively.
    Restrictions
    For Research Use only
  • Handling Advice
    The kit should not be used beyond the expiration date.
    Storage
    4 °C/-20 °C
    Storage Comment
    Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened EIA Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
  • Yang, Tsai, Chang, Chuang, Hsu, Chiang: "The interaction between circulating complement proteins and cutaneous microvascular endothelial cells in the development of childhood Henoch-Schönlein Purpura." in: PLoS ONE, Vol. 10, Issue 3, pp. e0120411, (2015) (PubMed).

    Sei, Mizuno, Suzuki, Imai, Higashide, Harris, Sakata, Iguchi, Fujiwara, Kodera, Maruyama, Matsuo, Ito: "Expression of membrane complement regulators, CD46, CD55 and CD59, in mesothelial cells of patients on peritoneal dialysis therapy." in: Molecular immunology, Vol. 65, Issue 2, pp. 302-9, (2015) (PubMed).

  • Target See all C3 ELISA Kits
    C3 (Complement Component 3 (C3))
    Alternative Name
    Complement C3 (C3 Products)
    Synonyms
    AHUS5 ELISA Kit, ARMD9 ELISA Kit, ASP ELISA Kit, C3a ELISA Kit, C3b ELISA Kit, CPAMD1 ELISA Kit, AI255234 ELISA Kit, HSE-MSF ELISA Kit, Plp ELISA Kit, C3 ELISA Kit, C3-1 ELISA Kit, sb:cb26 ELISA Kit, si:dkey-76b14.4 ELISA Kit, wu:fi34b03 ELISA Kit, wu:fj86e11 ELISA Kit, C3d ELISA Kit, asp ELISA Kit, cpamd1 ELISA Kit, c3 ELISA Kit, complement C3 ELISA Kit, complement component 3 ELISA Kit, complement component c3a, duplicate 1 ELISA Kit, complement component 3 L homeolog ELISA Kit, complement component C3 ELISA Kit, complement C3-like ELISA Kit, C3 ELISA Kit, c3a.1 ELISA Kit, c3.L ELISA Kit, LOC443475 ELISA Kit, c3 ELISA Kit, c3-1 ELISA Kit, LOC100060539 ELISA Kit
    Background
    Complement protein C3 is the fourth component to attach in the complement reaction sequence. It is a beta-globulin with a sedimentation coefficient of 5.5 and a molecular weight of 185,000. Complement C3 is a central molecule in the complement system whose activation is essential for all the important functions performed by this system. Complement C3 can promote phagocytosis during an inflammatory response against pathogens, but unregulated activation of complement C3 could lead to host cell damage. Low levels of serum complement C3 associates with hypocomplementaemia , primary biliary cirrhosis.
    Pathways
    Complement System, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Regulation of G-Protein Coupled Receptor Protein Signaling
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