SARS-CoV-2 N-Protein Antibody ELISA Kit ELISA Kit
- Human, SARS Coronavirus-2 (SARS-CoV-2)
- Detection Method
- Method Type
- Sandwich ELISA
- 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.
- Sample Type
- Nasal Secretions, Plasma, Saliva, Serum
- Analytical Method
- 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
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)
- 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.
- Reagent Preparation
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.)
- Sample Preparation
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.
- Assay Procedure
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.
- Bring all reagents to room temperature before use.
- 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!
- Add 45 µL sample dilution buffer to each sample well. Add 50 µL sample dilution buffer for blank well.
- 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.
- 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.
- Add 50 µLBiotin-labeled Antigento each well.Seal the plate with a cover and incubate at 37℃ for 30 min.
- 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.
- Add 50μl of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
- 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.
- 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.
- Add 50 μl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
- 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).
- Calculation of Results
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.
- For Research Use only
- 4 °C
- Storage Comment
- 2-8°C for 6 months
- Expiry Date
- 6 months