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SARS-CoV-2 Nucleocapsid ELISA Kit (SARS-CoV-2 N) ELISA Kit

SARS-CoV-2 N Reactivity: Human, SARS Coronavirus-2 (SARS-CoV-2) Colorimetric Sandwich ELISA 62.5 pg/mL - 4000 pg/mL Cell Culture Lysate
Catalog No. ABIN6952782
$681.85
Plus shipping costs $45.00
96 tests
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  • Target
    Reactivity
    Human, SARS Coronavirus-2 (SARS-CoV-2)
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Detection Range
    62.5 pg/mL - 4000 pg/mL
    Minimum Detection Limit
    62.5 pg/mL
    Application
    ELISA
    Purpose
    For quantitative detection of nucleoprotein in serum, plasma, tissue homogenates, cell culture supernatant, cell culture lysate and other biological fluids(throat swab, nose swab).
    Sample Type
    Cell Culture Lysate, Cell Culture Supernatant, Nasal Secretions, Plasma, Serum, Throat Swab, Tissue Homogenate
    Analytical Method
    Quantitative
    Specificity
    SARS-CoV-2 Nucleoprotein
    Cross-Reactivity (Details)
    SARS-CoV-2: 100%
    SARS-CoV: 73%
    MERS: 26%
    H1N1: < 2%
    Sensitivity
    37.5 pg/mL
    Components
    • ELISA Microplate(Dismountable)
    • Lyophilized Standard
    • Sample/Standard Dilution Buffer
    • Biotin-labeled Antibody(Concentrated)
    • Antibody Dilution Buffer
    • HRP-Streptavidin Conjugate(SABC)
    • SABC Dilution Buffer
    • TMB Substrate
    • Stop Solution
    • Wash Buffer(25X)
    • Plate Sealer
    • Product Description
  • Target
    Alternative Name
    SARS-CoV-2 Nucleocapsid Protein
  • Comment

    Washing
    Manual: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material.
    Automatic: Aspirate all wells, and then wash plate with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking 1 minute. (Note: set the height of the needles; be sure the fluid can be sipped up completely)

    Plate
    Pre-coated
    Protocol
    This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Capture antibody (monoclonal) was precoated onto 96-well plates. And the biotin conjugated antibody(monoclonal) was used as detection antibodies. The standards, test samples and biotin conjugated detection antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of target can be calculated.
    Reagent Preparation

    Bring all reagents and samples to room temperature for 20 minutes before use.

    1. Wash Buffer: If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
      Dilute 30ml Concentrated Wash Buffer into 750ml Wash Buffer with deionized or distilled water. Put unused solution back at 2-8°C.
    2. Standards:
      • Add 0.3ml Sample Dilution Buffer into one tube(labeled as zero tube),transfer 0.3ml fromstandard tube (100ng/ml) to zero tube and mix them thoroughly.
      • Note: If the standard tube concentration higher than the range of the kit,please dilute it and labeled as zero tube.
      • Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the Sample Dilution Buffer int o each tube. Add 0.3ml of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3ml from 1st tube to 2nd tube and mix them thoroughly. Transfer 0.3ml from 2nd tube to 3rd tube and mix them thoroughly, and so on. Sample Dilution Buffer was used for the blank control.
    3. Preparation of HRP-labeled Antibody Working Solution: Prepare it within 30min before experiment.
      • Calculate required total volume of the working solution: 50ul / well × quantity of wells. (Allow 55-60ul more than the total volume.)
      • Dilute the HRP-detection antibody with AntibodyDilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl HRPlabeled antibody into 99μl Antibody Dilution Buffer.)

    Sample Collection
    Serum: Place whole blood sample at room temperature for 2 hours or put it at 2-8°C overnight and centrifugation for 20 minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.
    • Plasma: Collect plasma using (EDTA-Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8°C within 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, high cholesterol samples.
    • Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (the volume depends on the weight of the tissue. Normal, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS) with a glass homogenizer on ice. To further break the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5minutes at 5000×g to get the supernatant. The total protein concentration was determined by BCA kit and the total protein concentration of each pore sample should not exceed 0.3mg.
    • Cell Culture Supernatant: Centrifuge supernatant for 20 minutes at 1000×g at 2 - 8°C to remove insoluble impurity and cell debris. Collect the clear supernatant and carry out the assay immediately.
    • Cell Culture Lysate: Commercial RIPA kits are recommended to follow the instructions provided. Generally, 0.5ml RIPA lysis buffer would be appropriate to 2x106 cells, DNA must to be removed. The total protein concentration was determined by BCA kit and the total protein concentration of each pore sample should not exceed 0.3mg.
    • Other Biological Fluids :(throat swab ,nose swab)Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assay immediately.
    Note: Samples to be used within 5 days can be stored at 2-8°C , besides that, samples must be stored at -20°C (assay ≤1 month) or -80°C(assay≤2 months) to avoid loss of bioactivity and contamination. Avoid multiple freeze-thaw cycles. The hemolytic samples are not suitable for this assay.
    Sample Preparation

    The user should estimate the concentration of target protein in the test sample, and select a proper dilution factor to make the diluted target protein concentration fall in the optimal detection range of the kit. Dilute the sample with the provided dilution buffer, and several trials may be necessary. The test sample must be well mixed with the dilution buff er. And also standard curves and sample should be making in pre-experiment. If samples with very high concentrations, dilute samples with PBS first and then dilute the samples with Sample Dilution.

    The matrix components in the sample will affect the test results, which it need to be diluted at least 1/50 with Sample Dilution Buffer before testing!

    Assay Procedure

    When diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB Substrate for 30 min at 37 °C. It is recommended to plot a standard curve for each test.

    1. Set standard, test samples (diluted at least 1/2 with Sample Dilution Buffer), control (blank) wells on the pre-coated plate respectively, and then, records their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (blank) wells!
    2. Prepare Standards: Aliquot 100μl of zero tube, 1sttube, 2ndtube, 3rdtube, 4thtube, 5thtube, 6thtube and Sample Dilution Buffer (blank) into the standard wells.
    3. Add Samples: Add 100μl of properly diluted sample into test sample wells.
    4. Incubate: Seal the plate with a cover and incubate at 37°C for 90 minutes.
    5. Wash: Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time.
    6. Biotin-labeled Antibody: Add 100μl Biotin-labeled antibody working solution into above wells (standard, test sample and blank wells). Add the solution at the bottom of each well without touching the sidewall, cover the plate and incubate at 37°C for 60 minutes.
    7. Wash: Remove the cover, and wash plate 3 times with Wash Buffer, and let the Wash Buffer stay in the wells for 1-2 minute each time.
    8. HRP-Streptavidin Conjugate (SABC): Add 100μl of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
    9. Wash: Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minute each time.
    10. TMB Substrate: Add 90μl TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 minutes. (Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. You can terminate the reaction when apparent gradient appeared in standard wells.)
    11. Stop: Add 50μl Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solution should be as the same as the TMB Substrate Solution.
    12. OD Measurement: Read the O.D. absorbance at 450 nm in Microplate Reader immediately after adding the stop solution.

    Calculation of Results

    Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of blank well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The target concentration of the samples can be interpolated from the standard curve. It is recommended to use some professional software to do this calculation, such as Curve Expert 1.3 or 1.4.
    Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.

    Assay Precision
    Intra-Assay: CV<8%
    Inter-Assay: CV<10%
    Restrictions
    For Research Use only
  • Storage
    4 °C
    Storage Comment
    2-8°C for 6 months
    Expiry Date
    6 months
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