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FOXP3 ELISA Kit

FOXP3 Reaktivität: Human Colorimetric Sandwich ELISA 0.312-20 ng/mL Tissue Homogenate
Produktnummer ABIN578989
  • Target Alle FOXP3 ELISA Kits anzeigen
    FOXP3 (Forkhead Box P3 (FOXP3))
    Reaktivität
    • 5
    • 4
    • 3
    • 2
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    Human
    Nachweismethode
    Colorimetric
    Methodentyp
    Sandwich ELISA
    Detektionsbereich
    0.312-20 ng/mL
    Untere Nachweisgrenze
    0.312 ng/mL
    Applikation
    ELISA
    Verwendungszweck
    This immunoassay kit allows for the in vitro quantitative determination of human FOXP3 concentrations in tissue homogenates and other biological fluids.
    Proben
    Tissue Homogenate
    Analytische Methode
    Quantitative
    Spezifität
    This assay recognizes recombinant and natural human FOXP3.
    Kreuzreaktivität (Details)
    No significant cross-reactivity or interference was observed.
    Sensitivität
    The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
    Produktmerkmale
    Homo sapiens,Human,Forkhead box protein P3,Scurfin,FOXP3,IPEX,JM2
    Bestandteile
    Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instruction (1)
    Benötigtes Material
    Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.
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  • Probenmenge
    100 μL
    Plattentyp
    Pre-coated
    Protokoll
    The microtiter plate provided in this kit has been pre-coated with an antibody specific to FOXP3. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for FOXP3. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain FOXP3, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of FOXP3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    Aufbereitung der Reagenzien

    Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 20 ng/ml. Allow the standard to sit for about 10 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the highest standard (20 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/ml 20 10 5 2.5 1.25 0.625 0.312 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A or B (1:100), respectively.

    Probennahme
    Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 5~10 mL of 1X PBS and stored overnight at -20 °C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at -20 °C. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Note: Tissue homogenates and other biological fluids to be used within 7 days may be stored at 2-8, otherwise samples must stored at -20 ( 1 month) or -80 ( 2 months) 3 to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.
    Testdurchführung

    Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and 4 remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
    1. Add 100 of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for two hours at 37 .
    2. Remove the liquid of each well, don’t wash.
    3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
    4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for one hour at 37 .
    6. Repeat the aspiration/wash process for five times as conducted in step
    4. 7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 15 - 30 minutes at 37 . Protect from light.
    8. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
    Important Note:1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
    2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10μl for once pipetting.
    3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay. 5
    4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
    5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
    6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    7. Duplication of all standards and specimens, although not required, is recommended.
    8. Substrate Solution is easily contaminated. Please protect it from light.

    Ergebnisberechnung

    Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the FOXP3 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Handhabung
    1. The kit should not be used beyond the expiration date on the kit label.
    2. Do not mix or substitute reagents with those from other lots or sources.
    3. If samples generate values higher than the highest standard, further dilute the samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
    4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
    5. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.
    Lagerung
    4 °C/-20 °C
    Informationen zur Lagerung
    The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
  • Target Alle FOXP3 ELISA Kits anzeigen
    FOXP3 (Forkhead Box P3 (FOXP3))
    Andere Bezeichnung
    FOXP3 (FOXP3 Produkte)
    Synonyme
    foxp3 ELISA Kit, FOXP3 ELISA Kit, AIID ELISA Kit, DIETER ELISA Kit, IPEX ELISA Kit, PIDX ELISA Kit, XPID ELISA Kit, JM2 ELISA Kit, scurfin ELISA Kit, sf ELISA Kit, RGD1562112 ELISA Kit, forkhead box P3 ELISA Kit, forkhead box P3a ELISA Kit, forkhead box P3 L homeolog ELISA Kit, FOXP3 ELISA Kit, foxp3a ELISA Kit, foxp3 ELISA Kit, foxp3.L ELISA Kit, Foxp3 ELISA Kit
    Hintergrund
    Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators and functions as the master regulator in the development and function of regulatory T cells. Human FOXP3 genes contain 11 coding exons. Exon-intron boundaries are identical across the coding regions of the mouse and human genes. By genomic sequence analysis, the FOXP3 gene maps to chromosome Xp11.23. Foxp3 gene is mutated in the X-linked syndrome of immunodysregulation, polyendocrinopathy, and enteropathy (IPEX). These mutations were in the forkhead domain of FOXP3, indicating that the mutations may disrupt critical DNA interactions. In mice, a Foxp3 mutation (a frameshift mutation that result in protein lacking the forkhead domain) is responsible for 'Scurfy', an X-linked recessive mouse mutant that results in lethality in hemizygous males 16 to 25 days after birth. The discovery of Foxp3 as a specific marker of regulatory T cells has recently led to an explosion of research in the biological properties of regulatory T cells. In human disease, alterations in numbers of regulatory T cells, and in particular those that express Foxp3, are found in a number of conditions. For example, patients with tumours have a local relative excess of Foxp3 positive T cells which inhibits the body's ability to suppress the formation of cancerous cells. On the other hand, patients with autoimmune disease such as systemic lupus erythematosus have a relative dysfunction of Foxp3 positive cells. In animal studies, regulatory T cells that express Foxp3 are critical in the transfer of immune tolerance, so that hopefully in the future this knowledge can be used to prevent transplant graft rejection. The induction or administration of Foxp3 positive T cells has, in animal studies, led to marked reductions in disease severity in models of diabetes, multiple sclerosis, asthma, inflammatory bowel disease, thyroiditis and renal disease. These discoveries give hope that cellular therapies using foxp3 positive cells may, one day, help overcome these diseases. 2
    Gen-ID
    3005
    Pathways
    Chromatin Binding, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Activated T Cell Proliferation
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