Fibrinogen ELISA Kit

Catalog No. ABIN612694

Product Details Fibrinogen ELISA Kit

Target: Fibrinogen

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Minimum Detection Limit: 100 ng/mL

Application: ELISA

Purpose: The AssayMax Human Fibrinogen ELISA kit is designed for detection of human FBG in plasma

Brand: AssayMax

Sample Type: Plasma

Analytical Method: Quantitative

Components: FBG Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a murine antibody against FBG. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. FBG Standard: Human FBG in a buffered protein base (240 µg, lyophilized). 1 Biotinylated FBG: 1 vial, lyophilized. 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). 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 pipettes). Deionized or distilled reagent grade water.

Application Details

Sample Volume: 25 μL

Assay Time: < 3 h

Plate: Pre-coated

Protocol: This assay employs a quantitative competitive enzyme immunoassay technique that measures FBG in less than 3 hours. A murine antibody specific for FBG has been pre-coated onto a 96- well microplate with removable strips. FBG in standards and samples is competed by a biotinylated FBG 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. 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 240 g of FBG Standard with 2 ml of MIx Diluent to generate a stock solution of 120 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 (120 g/ml) 1:3 with MIx Diluent to produce 40, 13.33, 4.44, 1.48, 0.49, and 0.16 g/ml solutions. MIx Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [FBG] ( g/ml) 1 part Standard (120 g/ml) + 2 parts MIx Diluent P1 40.00 P2 1 part P1 + 2 parts MIx Diluent 13.33 P3 1 part P2 + 2 parts MIx Diluent 4.44 P4 1 part P3 + 2 parts MIx Diluent 1.48 P5 1 part P4 + 2 parts MIx Diluent 0.49 P6 1 part P5 + 2 parts MIx Diluent 0.16 P7 MIx Diluent 0.00 Biotinylated FBG (2x): Dilute Biotinylated FBG with 2 ml MIx 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 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: 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 use supernatants for assay. Dilute samples 1: 2000 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA can also be used as anticoagulant).

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 FBG to each well (on top of the Standard or sample). 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 the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 8 minutes or till the optimal 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 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 5.1 % and 7.5% respectively.

Restrictions: For Research Use only

Handling

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 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.

References for Fibrinogen ELISA Kit (ABIN612694)

Chen, Wang, Yang, Li, Zhao, Wang, Yang, Wang: "MiR-320a contributes to atherogenesis by augmenting multiple risk factors and down-regulating SRF." in: Journal of cellular and molecular medicine, Vol. 19, Issue 5, pp. 970-85, (2015) (PubMed).

Target Details for Fibrinogen

Target: Fibrinogen

Alternative Name: Fibrinogen (FBG) (Fibrinogen Products)

Background: Fibrinogen (FBG) is a homodimer of molecular mass 340 kDa, made up of two sets of polypeptide chains, and synthesized in the parenchymal cell of the hepatocyte and in the megakaryocyte. FBG plays a major role in coagulation, and both elevated and decreased levels have clinical significance. Upon cleavage by thrombin in the initial stages of coagulation activation, FBG self-assembles to yield a fibrin clot matrix that subsequently is crosslinked by factor xIIIa to form an insoluble network. FBG also binds to the platelet glycoprotein IIbIIIa receptor so as to form bridges between platelets, thus facilitating aggregation. Elevated plasma FBG has been identified as an independent risk factor for coronary atherosclerosis and ischemic heart disease. Individuals with congenital absence of FBG, termed afibrinogenemia, have prolonged bleeding times.