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SARS-CoV-2 N-Protein Antibody ELISA Kit

Reaktivität: Human, SARS Coronavirus-2 (SARS-CoV-2) Colorimetric Sandwich ELISA Nasal Secretions, Plasma, Saliva, Serum
Produktnummer ABIN6952781
  • Target
    SARS-CoV-2 N-Protein Antibody
    Reaktivität
    Human, SARS Coronavirus-2 (SARS-CoV-2)
    Nachweismethode
    Colorimetric
    Methodentyp
    Sandwich ELISA
    Applikation
    ELISA
    Verwendungszweck
    anti-2019-nCoV IgG ELISA Kit allows for the in vitro qualitative determination of 2019 nCoV-Ig antibody in serum, plasma and saliva and nasal fluid.
    Proben
    Nasal Secretions, Plasma, Saliva, Serum
    Analytische Methode
    Qualitative
    Bestandteile
    • Coated assay plate
    • Negative Control (Ready-to-use)
    • Positive Control (Ready-to-use)
    • Sample Dilution Buffer
    • Biotin-conjugated Nucleocapsid (Concentrated)
    • Antigen Dilution Buffer
    • HRP-Streptavidin Conjugate(SABC)
    • SABC Dilution Buffer
    • Wash Buffer (25 x concentrate)
    • TMB Substrate
    • Stop solution
    • Plate Sealer
    • Product Description
  • Kommentare

    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)

    Plattentyp
    Pre-coated
    Protokoll
    This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Recombinant 2019 nCoV Nucleocapsid protein (antigen) was pre-coated onto 96-well plates. The Controls, test samples and Biotin-labeled antigen were added to the wells subsequently, and wash with wash buffer. HRP-Streptavidin Conjugate was added and unbound conjugates were washed away with wash buffer. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The optical density of developed color is read with a suitable photometer at 450nm with a selected reference wavelength within 650 nm.
    Aufbereitung der Reagenzien

    Wash Buffer Preparation:
    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.
    Preparation of HRP-conjugated anti-human IgG Working Solution:
    Prepare it within 1 hour 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-conjugated anti-human IgG with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl HRP-conjugated anti-human IgG into 99μl Antibody Dilution Buffer.)
    Aufbereitung der Proben
    • It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
    • If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
    • If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
    • Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).
    Note:
    Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20℃ for long term. Avoid multiple freeze-thaw cycles.
    • Serum: Coagulate the serum at room temperature (about 1 hour). Centrifuge at approximately 1000 × g for 15 min. Analyze the serum immediately or aliquot and store at -20℃.
    • Plasma: Collect plasma with heparin or EDTA as the anticoagulant. Centrifuge for 15min at 2-8°C at 1500 x g within 30 min of collection. For eliminating the platelet effect, suggesting that further centrifugation for 10 min at 2-8°C at 10000 x g. Analyze immediately or aliquot and store frozen at -20℃.
    • Saliva & Nasal fluid: Centrifuge samples for 20 minutes at 10000×g at 2-8°C. Collect supernatant and carry out the assay immediately.
    Testdurchführung

    AssayProcedure: 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.

    1. Bring all reagents to room temperature before use.
    2. Label the sample wells, 2 Negative Controls, 2 Positive Controls and 1 blank well.Wash plate 2 times before adding sample and control (blank) wells!
    3. Add 45 µL sample dilution buffer to each sample well. Add 50 µL sample dilution buffer for blank well.
    4. Add 5μL sample to each sample well. Add 50ul Negative Controls and Positive Controls to set Controls well and gently tap the plate to ensure thorough mixing. Seal the plate with a cover and incubate at 37℃ for 30 min.
    5. Remove the cover, and wash plate 2 times with Wash buffer and each time let the wash buffer stay in the wells for 1-2 min.
    6. Add 50 µLBiotin-labeled Antigento each well.Seal the plate with a cover and incubate at 37℃ for 30 min.
    7. Remove the cover, and wash plate 3 times with Wash buffer and each time let the wash buffer stay in the wells for 1-2 min.
    8. Add 50μl of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
    9. Remove the cover, and wash plate 5 times with Wash buffer and each time let the wash buffer stay in the wells for 1-2 min.
    10. Add 50μl of TMB substrate into each well. Gently tap the plate to ensure thorough mixing. Cover the plate and incubate at 37℃ in dark within 10-20 min. And the shades of obvious blue can be seen in the Positive Controls. Blank well wells show no obvious color.
    11. Add 50 μl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
    12. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution(Use the blank well to set zero).

    Ergebnisberechnung

    Cutoff Value =NCx × 2.1
    NCx: Mean Absorbance of Negative Control (when NCx<0.05, Calculate as 0.05).
    PCx : Mean Absorbance of Positive Control
    1. Sample with absorbance values < Cutoff Value are considered negative.
    Sample with absorbance value ≥ Cutoff Value are considered positive.
    2. PCx≤0.5, the test is regarded as Invalid, should be tested again.

    Beschränkungen
    Nur für Forschungszwecke einsetzbar
  • Lagerung
    4 °C
    Informationen zur Lagerung
    2-8°C for 6 months
    Haltbarkeit
    6 months
  • Target
    SARS-CoV-2 N-Protein Antibody
    Substanzklasse
    Antibody
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