Anti-SARS-CoV-2 IgG ELISA Kit (NP Version)
Background
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; previously provisionally named 2019 novel coronavirus or 2019-nCoV) disease (COVID-19) in Wuhan, China at the end of 2019 has caused a large global outbreak and is a major public health issue. SARS-CoV-2 is closely related to two bat-derived severe acute respiratory syndrome-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21. It is spread by human-to-human transmission via droplets or direct contact, and infection has been estimated to have mean incubation period of 6.4 days and a basic reproduction number of 2.24-3.58. Among patients with pneumonia caused by SARS-CoV-2, fever was the most common symptom, followed by cough. Bilateral lung involvement with ground-glass opacity was the most common finding from computed tomography images of the chest. Nucleocapsid protein (NP) is the most abundant protein of the coronavirus nucleocapsid containing the entire genomic RNA. Anti-NP antibodies have been shown to be the earliest and the most predominant antibodies detectable in patient’s blood samples after coronavirus infection.
Assay Principle
The Anti-SARS-CoV-2 IgG ELISA Kit (NP Version) is developed for the detection of IgG antibody against 2019-nCoV in human serum samples. In this ELISA format, recombinant neucleocapsid protein (NP) is pre-coated onto EIA plate, the diluted samples are added to their corresponding wells for antibody binding. After a subsequent incubation and wash steps, biotinylated anti-human IgG and Streptavidin-HRP are added to each well to bind the Fc region of captured IgG antibody sequentially, and catalyze a colorimetric reaction upon TMB addition at 450nm to quantitively determine the amount of anti-2019-nCoV IgG in human serum samples.
Intended Use
The Anti-SARS-CoV-2 IgG ELISA Kit is an indirect enzyme-linked immunoassay (ELISA) for detection of human IgG antibody against COVID-19 virus in human serum.
Kit Components
NP-Coated Microtiter Plate (CTG-EI061G1), 8-well strip×12
Biotinylated Anti-human IgG (CTG-EI061G2), 100 µl, 100X
Streptavidin-HRP, 100 µl, 100X
Sample Diluent, 30 ml, 1X
20×Wash Buffer, 50ml, 1X
Positive Control (CTG-EI061G3), 0.5ml, 1X
Negative Control, 0.05ml, 100X
TMB Substrate, 11ml, 1X
Stop Solution, 6ml, 1X
Sealing Tape, 4X
Instructions for Use, 1X
Materials Required but Not Supplied
The following materials and equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
• Microplate reader able to measure absorbance at 450 nm
• Micropipettes with capability of measuring volumes ranging from 1 μL to 1 mL
• Deionized or sterile water.
• Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer.Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer.
• Incubator and shaker.
Reagent Preparation
- Take out all kit reagents and required pre-coated strips and keep them at room temperature for 20 minutes.
- Biotinylated mouse anti-human IgG: Dilute the mouse anti-human IgG by 100 times using sample diluent.
- Strepavidin-HRP conjugate: Dilute the streptavidin-HRP conjugate by 100 times using sample diluent.
- Wash Buffer (1x): Prepare 1xWash buffer by mixing the 20xWash buffer (50ml) with 950ml of distilled water or deionized water. If precipitates are observed in the 20xWash buffer bottle, warm the bottle in a 37°C water batch until the precipitates disappear. The 1xWash buffer may be stored at 2-8°C for up to one month.
Sample Preparation
Test samples are suggested to be assayed immediately after separation of serum or plasma, or preferably stored frozen (-20°C or below) in aliquots. Avoid freeze-thaw cycles. Duplicate test is recommended.
Serum or plasma specimens with EDTA, sodium citrate or heparin can be tested. Highly lipaemic, icteric, or hemolytic specimens are not recommended. Specimens with visible microbial contamination should not be used. If samples are cloudy and containing particles, vortex serum or plasma samples to ensure homogeneity, then centrifuge at 10,000 to 15,000 rpm for 5 minutes to remove pellet.
Serum or plasma sample is generally required a 100-fold dilution in the 1xAssay buffer. A suggested dilution step is to add 10 μl of sample to 990 μl of 1 x Sample diluent.
Assay Procedure
- Bring all kit reagents and required pre-coated strips to room temperature. Positive, negative controls and blank must be assayed in duplicate.
- Apply 100μl/well of sample diluent, and then pipette 1μl of each sera sample (or its serial diluted samples, eg. 1:2, 1:4, 1:8…), and negative control to the plate. Vortex gently.
- Apply 100μl/well of sample diluents (blank) and positive control, respectively, to their corresponding wells.
- Cover the plate with sealing tape, and incubate the plate at 37℃ for 60 min in an incubator.
- After the incubation, wash the plate 5 times with a microplate washer and tip off the residuals.
- Add 100μl of 1X biotinylated anti-human IgG to the corresponding wells, and incubate the plate at 37℃ for 30min.
- Wash the plate as step 6 and tip off the residuals.
- Add 100μl of 1X Streptavidin-HRP conjugate to the corresponding wells, and incubate the plate at 37℃ for 30min.
- Wash the plate as step 6 and tip off the residuals.
- Add 100μl/well of TMB substrate into all wells. Incubate the plate at room temperature in dark for exact 15 min.
- After the incubation, add 50μl/well of stop solution into all wells, and mix thoroughly.
- Read the value of optical density at 450nm with a microplate spectrophotometer.
Quality Control
Each microplate should be considered separately when calculating and interpreting the results of the assay, regardless of the number of plates concurrently processed. The test results are valid if the Quality Control criteria are fulfilled. It is recommended that each laboratory must establish appropriate quality control system with quality control material similar to or identical with the patient specimen being analyzed.
Typical Results
Sample | OD450nm |
Negative Control | 0.089 |
0.110 | |
0.093 | |
Serum from COVID-19 patients | 1.522 |
1.716 | |
1.294 |
Intra-assay (Within-Run): CV = 6.2%
Inter-assay (Run-to-Run): CV = 7.3%
Notes
1. This kit is for research only, not for clinical diagnosis application.
2. This kit should be kept at 4℃. Once opened up, all the contents in this kit should be used as soon as possible to avoid any possible microorganism contamination.
3. Ensure the pipetting sample volume is correct.
4. Ensure time and temperature of assay incubation are correct.
5. Materials included in this kit should not be used past the expiration date on the kit label.
6. Assay results are not definitive for the diagnosis of nCoV infection, and cannot be used as the sole basis of patient management decision and must be combined with clinical observations, patient history, epidemiological information, and other laboratory evidences.
7. Please read the whole manual before performing the experiment.
Storage Instruction
The kit is stable until the expiring date only when stored at 2-8°C in sealed foil pouches. The kit should be stored at 2-8°C upon receipt, and all reagents should be equilibrated to room temperature before use. Remove any unused antigen-coated strips from the microplate, return them to the foil pouch and re-seal. Once opened, the strips may be stored at 2-8°C for up to one month. To assure maximum performance, protect the reagents from contamination with microorganism or chemicals during storage.
Procedure Summary
Add 100 μl of sample to each well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well five times.
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Add 100 μl of Biotinylated detection antibody solution to each well.
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Incubate at room temperature for 30 min.
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Aspirate and wash each well five times.
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Add 100 μl of Streptavidin-HRP solution to each well.
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Incubate at room temperature for 30 min.
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Aspirate and wash each well five times.
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Add 100 μl of TMB Substrate to each well.
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Incubate at room temperature for 15 minutes.
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Add 50 μl of Stop solution to each well.
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Measure absorbance of each well at 450 nm.
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Calculation and Interpretation
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