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Human Neurofilament, Light Polypeptide (NEFL) Sandwich ELISA Kit - 96 wells plate

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    Human Neurofilament, Light Polypeptide (NEFL) ELISA Kit is an ELISA Kit for the in vitro quantitative measurement of Human NEFL concentrations in serum, plasma, cerebrospinal fluid (csf) and other biological fluids. This assay has high sensitivity and excellent specificity for detection of NEFL
    No significant cross-reactivity or interference between NEFL and analogues was observed. 


    IntroductionNeurofilament light polypeptide (NFL), also known as neurofilament light chain, is a neurofilament protein that in humans is encoded by the NEFL gene. Neurofilament light chain is a biomarker that can be measured with immunoassays in cerebrospinal fluid and plasma and reflects axonal damage in a wide variety of neurological disorders. It is a useful marker for disease monitoring in amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, and more recently Huntington's disease.
    TargetNeurofilament, Light Polypeptide (NEFL)
    ReactivityHuman
    Tested ApplicationsELISA
    Recommended dilutionsOptimal dilutions/concentrations should be determined by the end user.
    StorageShipped at 4 °C. Upon receipt, store the kit according to the storage instruction in the kit's manual.
    ValidityThe validity for this kit is 6 months.
    StabilityThe stability of the kit is determined by the rate of activity loss. The loss rate is less than 5% within the expiration date under appropriate storage conditions. To minimize performance fluctuations, operation procedures and lab conditions should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same user throughout.
    Test Range15.6 pg/ml - 1000 pg/ml
    Sensitivity< 5.7 pg/ml
    Standard FormLyophilized
    Detection MethodColorimetric
    Assay TypeSandwich
    Assay DataQuantitative
    Sample TypeSerum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids.
    Target TypeAntigen
    Assay PrincipleThis kit is based on sandwich enzyme-linked immuno-sorbent assay technology. An antibody is pre-coated onto a 96-well plate. Standards, test samples, and biotin-conjugated reagent are added to the wells and incubated. The HRP-conjugated reagent is then added, and the whole plate is incubated. Unbound conjugates are removed using wash buffer at each stage. TMB substrate is used to quantify the HRP enzymatic reaction. After TMB substrate is added, only wells that contain sufficient NEFL will produce a blue coloured product, which then changes to yellow after adding the acidic stop solution. The intensity of the yellow colour is proportional to the NEFL amount bound on the plate. The Optical Density (OD) is measured spectrophotometrically at 450 nm in a microplate reader, from which the concentration of NEFL can be calculated.
    Kit ComponentsThe kit components listed are for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
    • Pre-coated 96-Well Microplate
    • Standard
    • Standard Diluent Buffer
    • Wash Buffer
    • Detection Reagent A
    • Detection Reagent B
    • Diluent A
    • Diluent B
    • TMB Substrate
    • Stop Solution
    • Plate Sealer
    Material Required But Not Provided
    • 37°C incubator
    • Multi and single channel pipettes and sterile pipette tips
    • Squirt bottle or automated microplate washer
    • 1.5 ml tubes
    • Distilled water
    • Absorbent filter papers
    • 100 ml and 1 liter graduated cylinders
    • Microplate reader (wavelength: 450 nm)
    • ELISA Shaker
    Reagent PreparationThis procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
    • 1) Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the Standard Diluent buffer to carry out serial dilutions of the standard solution, as instructed in the Protocol.
    • 2) Wash Buffer: Dilute the concentrated Wash Buffer with distilled water, as instructed in the Protocol.
    • 3) Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, at 1:100.
    Assay ProcedureThis procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
    • 1) Set standard, test samples and control wells.
    • 2) Aliquot 100 µl of diluted standard into the standard wells.
    • 3) Aliquot 100 µl of Standard Diluent buffer into control (zero) well.
    • 4) Aliquot 100 µl of diluted samples into the sample wells. Incubate for 1 hr at 37 °C.
    • 5) Aliquot 100 µl of Detection Reagent A to each well. Incubate for 1 hr at 37 °C.
    • 6) Wash 3 times.
    • 7) Aliquot 100 µl of Detection Reagent B to each well. Incubate for 30 mins at 37 °C.
    • 8) Wash 5 times.
    • 9) Aliquot 90 µl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C.
    • 10) Aliquot 50 µl of Stop Solution.
    • 11) Measure the OD at 450 nm.
    ProtocolThis procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product.
    • Equilibrate the kit components and samples to room temperature (18 - 25 °C) before use. It is recommended to plot a standard curve for each test.
    • 1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample at least in duplicate.
    • 2. Add 100 µL of each standard, control and sample into the appropriate wells. Seal the plate with a cover and incubate for 1 h at 37°C.
    • 3. Remove the cover and discard the liquid.
    • 4. Add 100 µl of the detection Reagent A working solution to each well. Seal the plate with a cover and incubate for 1 h at 37°C.
    • 5. Remove the cover and discard the solution. Wash the plate 3 times with 1X Wash Buffer.
    • 6. Add 100 µL of Detection Reagent B working solution into each well, seal and incubate at 37°C for 30 min.
    • 7. Discard the solution and wash the plate 5 times with wash buffer as explained in previous step.
    • 8. Aliquot 90 µl of TMB Substrate into each well. Seal the plate with a cover and incubate at 37°C for 10-20 min. Avoid exposure to light. The incubation time is for reference use only, the optimal time should be determined by end user. Do not exceed 30 min.
    • 9. Add 50 µL of Stop Solution to each well. Read at 450 nm immediately.
    Results CalculationFor calculation, average the O.D.450 duplicate readings for each reference standard and each sample and substract the average control (zero) O.D.450 reading. The standard curve can be plotted as the relative O.D.450 of each reference standard solution [Y] vs. the respective concentration of each standard solution (X). The NEFL concentration of the samples can be interpolated from the standard curve.
    Assay PrecisionIntra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of NEFL were were tested 20 times on one plate, respectively.

    Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of NEFL were tested on 3 different plates, 8 replicates in each plate.

    CV (%) = (Standard Deviation / mean) × 100

    Intra-Assay: CV<10%

    Inter-Assay: CV<12%
    AvailabilityShipped within 5-7 working days.
    NoteThis product is for research use only.

    The range and sensitivity is subject to change. Please contact us for the latest product information. For accurate results, sample concentrations must be diluted to mid-range of the kit. If you require a specific range, please contact us in advance or write your request in your order comments.

    Please note that our ELISA and CLIA kits are optimised for detection of native samples, rather than recombinant proteins. We are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native protein.
    Standard10000 pg/ml