Fibrinogen ELISA Kit

Catalog No. ABIN612683

Product Details Fibrinogen ELISA Kit

Target: Fibrinogen

Reactivity: Dog

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Minimum Detection Limit: 12 ng/mL

Application: ELISA

Purpose: The AssayMax Canine Fibrinogen ELISA kit is designed for detection of canine FBG in urine and cell culture media

Brand: AssayMax

Sample Type: Cell Culture Supernatant

Analytical Method: Quantitative

Components: FBG Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal 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: Canine FBG in a buffered protein base (800 ng, lyophilized).). Biotinylated Canine FBG Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against canine FBG (80µl). EIA 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

Application Details

Sample Volume: 50 μL

Assay Time: < 4 h

Plate: Pre-coated

Protocol: This assay employs a quantitative sandwich enzyme immunoassay technique that measures canine FBG in less than 4 hours. A polyclonal antibody specific for canine FBG has been pre-coated onto a 96-well microplate with removable strips. FBG in standards and samples is sandwiched by the immobilized polyclonal antibody and biotinylated polyclonal antibody specific for canine FBG, 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. 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 800 ng of FBG Standard with 1 ml of EIA Diluent to generate a standard solution of 800 ng/ml. Prepare duplicate or triplicate standard points by serially diluting the standard solution (800 ng/ml) twofold with EIA Diluent to produce 400, 2 200, 100, 50, 25, and 12.5 ng/ml solutions. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [FBG] (ng/ml) P1 1 part Standard (800 ng/ml) 800.00 P2 1 part P1 + 1 part EIA Diluent 400.00 P3 1 part P2 + 1 part EIA Diluent 200.00 P4 1 part P3 + 1 part EIA Diluent 100.00 P5 1 part P4 + 1 part EIA Diluent 50.000 P6 1 part P5 + 1 part EIA Diluent 25.00 P7 1 part P6 + 1 part EIA Diluent 12.50 P8 EIA Diluent 0.000 Biotinylated Canine FBG Antibody (100x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:100 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: Urine: Collect urine using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:4 with EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Media: 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.

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 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 Canine FBG 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 20 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 4.9 % and 7.1% respectively.

Restrictions: For Research Use only

Handling

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 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.

Target Details for Fibrinogen

Target: Fibrinogen

Alternative Name: Fibrinogen (FBG) (Fibrinogen Products)

Synonyms: fib2 ELISA Kit, MGC53620 ELISA Kit, fibrinogen ELISA Kit, sb:cb892 ELISA Kit, wu:fa55c12 ELISA Kit, wu:fb20e08 ELISA Kit, wu:fb59b03 ELISA Kit, wu:fi39d04 ELISA Kit, zgc:92117 ELISA Kit, fibrinogen alpha chain ELISA Kit, fibrinogen gamma chain ELISA Kit, fga ELISA Kit, FGA ELISA Kit, LOC698244 ELISA Kit, CpipJ_CPIJ010090 ELISA Kit, FGG ELISA Kit

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.