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MIF ELISA Kit

MIF Reactivity: Human Colorimetric Sandwich ELISA 0.313-20 ng/mL Cell Culture Cells, Milk, Plasma, Saliva, Serum
Catalog No. ABIN5564627
  • Target See all MIF ELISA Kits
    MIF (Macrophage Migration Inhibitory Factor (Glycosylation-Inhibiting Factor) (MIF))
    Reactivity
    • 5
    • 4
    • 3
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    Human
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    0.313-20 ng/mL
    Minimum Detection Limit
    0.313 ng/mL
    Application
    ELISA
    Purpose
    The AssayMax™ Macrophage Migration Inhibitory Factor (MIF) ELISA (Enzyme- Linked Immunosorbent Assay) kit is designed for detection of human MIF in plasma, serum, saliva, milk, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human MIF in 4 hours. A polyclonal antibody specific for human MIF has been pre- coated onto a 96-well microplate with removable strips. MIF in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for MIF, which is recognized by a streptavidin- peroxidase 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. 2
    Brand
    AssayMax™
    Sample Type
    Cell Culture Cells, Milk, Plasma, Saliva, Serum
    Analytical Method
    Quantitative
    Components
    Human MIF Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human MIF. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human MIF Standard: Human MIF in a buffered protein base (10 ng, lyophilized). Biotinylated Human MIF Antibody (40x): A 40-fold concentrated biotinylated polyclonal antibody against MIF (150 l). 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 (80 l). 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 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)
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  • Assay Time
    4 h
    Plate
    Pre-coated
    Protocol
    • Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
    • Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
    • Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
    • Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 30 minutes.
    • Step 5. 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. 4 Dilute the MIX Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °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. Dilute plasma samples 1:2 with MIX Diluent and assay. 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 3000 x g for 10 minutes, and remove serum. Dilute serum samples 1:2 with MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute saliva samples 1:2 into MIX Diluent and assay. The undiluted samples can be stored at -20 ° C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute milk samples 1:20 into MIX Diluent and assay. The undiluted samples can be stored at -20 ° C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store the remaining samples at -20 °C or below. Avoid repeated freeze-thaw cycles.
    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. 5 Add 50 l of Human MIF Standard or sample per well. 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. Add 50 l of Biotinylated Human MIF Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l 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 l of Chromogen Substrate per well and incubate for 30 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 l 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 (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 Advice
    This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (working diluent buffer, wash buffer, standard, 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 insert. 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 Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.
    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 and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. 3 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 at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
  • Target See all MIF ELISA Kits
    MIF (Macrophage Migration Inhibitory Factor (Glycosylation-Inhibiting Factor) (MIF))
    Alternative Name
    Macrophage Migration Inhibitory Factor (MIF) (MIF Products)
    Synonyms
    mif ELISA Kit, Mif ELISA Kit, gif ELISA Kit, glif ELISA Kit, mmif ELISA Kit, LOC100136498 ELISA Kit, LOC100284350 ELISA Kit, LOC100284546 ELISA Kit, GIF ELISA Kit, Glif ELISA Kit, GLIF ELISA Kit, MMIF ELISA Kit, macrophage migration inhibitory factor L homeolog ELISA Kit, macrophage migration inhibitory factor ELISA Kit, macrophage migration inhibitory factor (glycosylation-inhibiting factor) ELISA Kit, mif.L ELISA Kit, mif ELISA Kit, MIF ELISA Kit, Mif ELISA Kit, PHATRDRAFT_49660 ELISA Kit, LOC100136498 ELISA Kit, cl405_1 ELISA Kit, LOC100284546 ELISA Kit
    Background
    Macrophage migration inhibitory factor (MIF), also known as glycosylation- inhibiting factor (GIF), L-dopachrome isomerase, and phenylpyruvate tautomerase, is a major secreted protein. It consists of 115-amino acids with a calculated molecular weight of 12.6 kDa (1). The MIF molecule is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet (2). As a proinflammatory cytokine, MIF is involved in the innate and adaptive immune responses. Present in most cells including pituitary cells, T cells, monocytes/macrophages, and epithelial cells, MIF is released upon glucocorticoid action, infection, and stress stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids to control both local and systemic immune responses. The binding of MIF to CD74, a histocompatibility complex class-II transmembrane protein, initiates signal transduction through mitogen-activated protein kinase (MAPK) cascade. In addition to endocrine and enzymatic functions, MIF plays a role as a mediator in regulating the function of macrophages in host defense (3-6).
    Gene ID
    4282
    UniProt
    P14174
    Pathways
    Regulation of Systemic Arterial Blood Pressure by Hormones, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Smooth Muscle Cell Migration, Negative Regulation of intrinsic apoptotic Signaling
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