Fibrinogen ELISA Kit

Catalog No. ABIN5564604

This Colorimetric ELISA kit is designed for the quantitative measurement of Human Fibrinogen.

Quick Overview for Fibrinogen ELISA Kit (ABIN5564604)

Target: Fibrinogen

Reactivity: Human

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 2.5-40 μg/mL

Application: ELISA

Sample Type: Plasma

Product Details Fibrinogen ELISA Kit

Minimum Detection Limit: 2.5 μg/mL

Purpose: The AssayMax™ Human Fibrinogen ELISA (Enzyme-Linked Immunosorbent Assay) Kit is designed for detection of FBG in human plasma samples. This assay employs a quantitative competitive enzyme immunoassay technique that measures human FBG in approximately 3 hours. A murine antibody specific for human FBG has been pre-coated onto a 96-well microplate with removable strips. FBG in standards and samples is competed with a biotinylated human FBG protein sandwiched by the immobilized antibody and streptavidin-peroxidase (SP) conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Analytical Method: Quantitative

Components: Human Fibrinogen Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a murine antibody against human FBG. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Fibrinogen Standard: Human FBG in a buffered protein base (160 g, lyophilized). Biotinylated Human Fibrinogen Protein (2x): 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). SP Conjugate (100x): A 100-fold concentrate (80 l). Chromogen Substrate (1x): A stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution (1x): A 0.5 N hydrochloric acid solution to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Application Details

Assay Time: 3 h

Plate: Pre-coated

Protocol:

• Step 1. Add 25 μL of Standard or Sample and 25 μL of Biotinylated Protein per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 3. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 12 minutes.
• Step 4. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human Fibrinogen Standard: Reconstitute the Human Fibrinogen Standard (160 g) with 4 mL of MIX Diluent to generate a 40 g/mL standard stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution (40 g/mL) 2-fold with equal volume of MIX Diluent to produce 20, 10, 5, and 2.5 g/mL solutions. MIX Diluent serves as the zero standard (0 g/mL). Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. 4 Standard Point Dilution [FBG] (μg/mL) P1 1 part Standard (40 g/mL) 40 P2 1 part P1 + 1 part MIX Diluent 20 P3 1 part P2 + 1 part MIX Diluent 10 P4 1 part P3 + 1 part MIX Diluent 5.0 P5 1 part P4 + 1 part MIX Diluent 2.5 P6 MIX Diluent 0.0 Biotinylated Human Fibrinogen Protein (2x): Reconstitute the Biotinylated Human Fibrinogen Protein with 4 mL of MIX Diluent to produce a stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to making dilution. From the stock solution, dilute 2-fold with MIX Diluent to produce a 1x working solution. Any remaining stock solution should be stored at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent to produce a 1x solution. The undiluted conjugate should be stored at -20 °C.

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 and collect plasma. A 500-fold sample dilution is suggested into MIX Diluent, however, user should determine optimal dilution factor depending on application needs. 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).

Assay Procedure:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °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 25 l of Human Fibrinogen Standard or sample to each well and immediately add 25 l of Biotinylated Human Fibrinogen Protein to each well (on top of the standard or sample). Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l 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 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. 5 Add 50 l of SP Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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 l of Chromogen Substrate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 12 minutes or until the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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 low 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 (OD) 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

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated protein, 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 insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date. 2

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate 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 Standard and Biotinylated Protein at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Target Details for Fibrinogen

Target: Fibrinogen

Alternative Name: Fibrinogen (FBG)

Background: Fibrinogen (FBG) is a homodimer (340 kDa) that is made up of two sets of alpha, beta, and gamma polypeptide chains. FBG is synthesized in the parenchymal cell of the hepatocyte and in the megakaryocyte (1). FBG plays a major role in coagulation. Upon cleavage by thrombin in the initial stages of coagulation activation, FBG self-assembles to yield a fibrin clot matrix that subsequently is cross-linked by factor XIIIa to form an insoluble network. FBG also binds to the platelet glycoprotein IIbIIIa receptor to form bridges between platelets, thus facilitating aggregation (2).

Gene ID: 2243, 2244, 2266

UniProt: P02671, P02675, P02679