Complement Component 3 (C3) ELISA Kit

Details for Product No. ABIN612678
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Target Name (Antigen)
Synonyms C3, C3-1, sb:cb26, si:dkey-76b14.4, wu:fi34b03, wu:fj86e11, asp, cpamd1, AI255234, ASP, HSE-MSF, Plp, AHUS5, ARMD9, C3a, C3b, CPAMD1, C3d, c3, c3_D
Reactivity
Alternatives Human
Kits with alternative reactivity to:
(11), (3), (9), (2), (8), (24), (8), (12), (11), (9), (12), (2), (1)
Methode Type Sandwich ELISA
Minimum Detection Limit 0.6 ng/mL
Application
ELISA
Pubmed 9 references available
Quantity 96 tests
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Catalog No. ABIN612678
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Purpose The AssayMax Human Complement C3 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human complement C3 in urine, milk, saliva and cell culture supernatants
Sample Type Cell Culture Supernatant
Detection Method Colorimetric
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 (3.2 µg, lyophilized). Biotinylated Complement C3 Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against Complement C3 (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). 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
Alternative Name Complement C3
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.
Research Area Complement Factors
Sample Volume 50 μL
Assay Time < 4 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique that measures human complement C3 in less than 4 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 sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for Complement C3, which is recognized by a 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. MIx Diluent Concentrate (10x): Dilute the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 3.2 g of Complement C3 Standard with 5 ml of MIx Diluent to generate a solution of 640 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The stock standard (640 ng/ml) should be further diluted 1:16 with MIx to generate a standard solution of 40 ng/ml. Prepare duplicate or triplicate standard points by serially diluting the standard solution (40 ng/ml) 1:2 with MIx Diluent to produce 20, 10, 5, 2.5, 1.25 and 0.625 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Complement C3] (ng/ml) P1 Stock (640 ng/ml) + 15 part MIx Diluent 40.000 P2 1 part P1 + 1 part MIx Diluent 20.000 P3 1 part P2 + 1 part MIx Diluent 10.000 P4 1 part P3 + 1 part MIx Diluent 5.000 P5 1 part P4 + 1 part MIx Diluent 2.500 P6 1 part P5 + 1 part MIx Diluent 1.250 P7 1 part P6 + 1 part MIx Diluent 0.625 P8 MIx Diluent 0.000 Biotinylated Complement C3 Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 with MIx 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 MIx Diluent. Any remaining solution should be frozen at -20°C.
Sample Collection Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:8 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles Saliva: Collect saliva using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:100 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes and assay. Milk dilution is suggested at 1:2000 into MIx Diluent, however, the user should determine the optimal dilution factor. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. 2
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 50 µL of Complement C3 standard or sample per well. 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 Biotinylated Complement C3 Antibody to each well and incubate for one hour. Wash a microplate as described above. 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 10 minutes or till 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 4.8 % and 7.5 % respectively.
Restrictions For Research Use only
Handling Advice Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, and SP conjugate) 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. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx 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.
Product cited in: Hugli: "Human anaphylatoxin (C3a) from the third component of complement. Primary structure." in: The Journal of biological chemistry, Vol. 250, Issue 21, pp. 8293-301, 1976 (PubMed).

de Bruijn, Fey: "Human complement component C3: cDNA coding sequence and derived primary structure." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 82, Issue 3, pp. 708-12, 1985 (PubMed).

Morris, Goldberger, Colten et al.: "Biosynthesis and processing of a human precursor complement protein, pro-C3, in a hepatoma-derived cell line." in: Science (New York, N.Y.), Vol. 215, Issue 4531, pp. 399-400, 1982 (PubMed).

Tögel, Wiche, Propst: "Novel features of the light chain of microtubule-associated protein MAP1B: microtubule stabilization, self interaction, actin filament binding, and regulation by the heavy chain." in: The Journal of cell biology, Vol. 143, Issue 3, pp. 695-707, 1998 (PubMed).

Ma, Himes, Shea et al.: "Axonal transport of microtubule-associated protein 1B (MAP1B) in the sciatic nerve of adult rat: distinct transport rates of different isoforms." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 20, Issue 6, pp. 2112-20, 2000 (PubMed).

Wiesmann, Katschke, Yin et al.: "Structure of C3b in complex with CRIg gives insights into regulation of complement activation." in: Nature, Vol. 444, Issue 7116, pp. 217-20, 2006 (PubMed).

Janssen, Christodoulidou, McCarthy et al.: "Structure of C3b reveals conformational changes that underlie complement activity." in: Nature, Vol. 444, Issue 7116, pp. 213-6, 2006 (PubMed).

Ram, Lewis, Rice: "Infections of people with complement deficiencies and patients who have undergone splenectomy." in: Clinical microbiology reviews, Vol. 23, Issue 4, pp. 740-80, 2010 (PubMed).

Stokowska, Olsson, Holmegaard et al.: "Plasma C3 and C3a levels in cryptogenic and large-vessel disease stroke: associations with outcome." in: Cerebrovascular diseases (Basel, Switzerland), Vol. 32, Issue 2, pp. 114-22, 2011 (PubMed).

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