SARS-CoV-2 Nucleocapsid Protein ELISA Kit

SARS-CoV-2 Nucleocapsid Protein ELISA Kit

About COVID-19

SARS-CoV-2, the causative viral agent of the disease COVID-19, is a coronavirus which bears the transmembrane glycoprotein spikes (S protein) typical of viruses in its clade. These spikes are a prominent target of human immune responses and have been found to be highly immunogenic. The receptor-binding domain (RBD) of the S protein is particularly targeted by neutralising antibodies.

The spikes on SARS-CoV-2 allows the virus to enter host cells through the human receptor angiotensin converting enzyme 2 (ACE2), present in alveolar epithelial cells.

The time between initial viral exposure and symptom onset is known as the incubation period. For COVID-19, the average incubation period has been reported to be between five and six days. However, there is considerable variation in incubation time, with some studies suggesting symptoms can appear as soon as three days post-exposure or as late as thirteen days post-exposure.

Fever, fatigue, dry cough are the main symptoms. Some patients could present nasal congestion, runny nose, diarrhea and other symptoms. In severe cases, dyspnea occurred after one week which could lead to acute respiratory distress syndrome (ARDS), septic shock, refractory metabolic acidosis and coagulation dysfunction were rapidly advanced. Particularly, in the course of severe and critical cases, the fever can be moderate or low, and sometimes it is not even obvious. Some patients who showed only low fever, slight fatigue, and etc. without pneumonia manifestations, are able to recover in one week.

How does our SARS-CoV-2 Nucleocapsid Protein ELISA Kit work?

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to SARS-CoV-2 N protein. Samples (or Standards) and biotinylated detection antibody specific for SARS-CoV-2 N protein are added to the micro ELISA plate wells. SARS-CoV-2 N protein would combine with the specific antibody. Then Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away.

The substrate solution is added to each well. Only those wells that contain SARS-CoV-2 N protein, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 ± 2 nm. The OD value is proportional to the concentration of SARSCoV-2 N protein. You can calculate the concentration of SARS-CoV-2 N protein in the samples by comparing the OD of the samples to the standard curve.

SARS-CoV-2 Nucleocapsid Protein ELISA Kit Contents

Other materials required

• Microplate Reader with 450 nm wavelength filter or dual-wavelength (450/630 nm)
• High-precision transfer pettor, EP tubes and disposable pipette tips
• Incubator capable of maintaining 37„ƒ
• Deionized or distilled water
• Absorbent paper
• Loading slot for Wash Buffer

Sample collection and preparation

Serum: Allow samples to clot for 2 hours at room temperature or overnight at 2-8 „ƒ before centrifugation for 20 min at 1000×g at 2-8„ƒ. Collect the supernatant to carry out the assay. The suspended fibrous protein may cause a false positive result if not fully precipitated. Obviously contaminated samples can't be detected

Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2-8„ƒ within 30 min of collection. Collect the supernatant to carry out the assay.

Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells using trypsin. Collect the cell suspension into a centrifuge tube and centrifuge for 5 min at 1000×g. Discard the medium and wash the cells 3 times with pre-cooled PBS. For each 1×106 cells, add 150-250 μL of pre-cooled PBS to keep the cells suspended. Repeat the freeze-thaw process several times or use an ultrasonic cell disrupter until the cells are fully lysed. Centrifuge for 10min at 1500×g at 2-8„ƒ. Remove the cell fragments, collect the supernatant to carry out the assay

Cell culture supernatant or other biological fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8„ƒ. Collect the supernatant to carry out the assay.

Note for samples:

1. Tubes for blood collection should be disposable and be non-endotoxin. Samples with high hemolysis or much lipid are not suitable for ELISA assay.
2. Samples should be assayed within 7 days when stored at 2-8„ƒ, otherwise samples must be divided up and stored at -20„ƒ (≤1 month) or -80„ƒ (≤3 months). Avoid repeated freeze-thaw cycles. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates.
3. Please predict the concentration before assay. For tested samples, a preliminary experiment is strongly suggested to determine the optimal dilution factors for their particular experiments.
4. If the sample type is not included in the manual, a preliminary experiment is suggested to verify the validity.
5. If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance.
6. Some recombinant protein may not be detected due to a mismatching with the coated antibody or detection antibody.

Reagent Preparation

1. Bring all reagents to room temperature (18-25„ƒ) before use. If the kit will not be used up in one assay, please only take out the necessary strips and reagents for present experiment, and store the remaining strips and reagents at required condition.
2. Wash Buffer: Dilute 30mL of Concentrated Wash Buffer with 720mL of deionized or distilled water to prepare 750mL of Wash Buffer. Note: if crystals have formed in the concentrate, warm it in a 40„ƒ water bath and mix it gently until the crystals have completely dissolved.
3. Standard working solution: Centrifuge the standard at 10,000×g for 1 min. Add 1.0 mL of Reference Standard &Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 25ng/mL(or add 1.0mL of Reference Standard &Sample Diluent, let it stand for 1-2 min and then mix it thoroughly with a vortex meter of low speed. Bubbles generated during vortex could be removed by centrifuging at a relatively low speed). Then make serial dilutions as needed. The recommended dilution gradient is as follows: 25, 12.5, 6.25, 3.13, 1.56, 0.78 0.39, 0ng/mL.
   Dilution method: Take 7 EP tubes, add 500μL of Reference Standard & Sample Diluent to each tube. Pipette 500μL of the 25ng/mL working solution to the first tube and mix up to produce a 12.5ng/mL working solution. Pipette 500μL of the solution from the former tube into the latter one according to this step. The illustration below is for reference. Note: the last tube is regarded as a blank. Don’t pipette solution into it from the former tube.
4. Biotinylated Detection Ab working solution: Calculate the required amount before the experiment (100μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated Biotinylated Detection Ab at 800×g for 1 min, then dilute the 100× Concentrated Biotinylated Detection Ab to 1× working solution with Biotinylated Detection Ab Diluent(Concentrated Biotinylated Detection Ab: Biotinylated Detection Ab Diluent= 1: 99).
5. HRP Conjugate working solution: Calculate the required amount before the experiment (100μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the Concentrated HRP Conjugate at 800×g for 1 min, then dilute the 100× Concentrated HRP Conjugate to 1× working solution with HRP Conjugate Diluent(Concentrated HRP Conjugate: HRP Conjugate Diluent= 1:99).

SARS-CoV-2 Nucleocapsid Protein ELISA Kit Assay Procedure

1. Determine wells for diluted standard, blank and sample. Add 100μL each dilution of standard, blank and sample into the appropriate wells (It is recommended that all samples and standards be assayed in duplicate). Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37„ƒ. Note: solutions should be added to the bottom of the micro ELISA plate well, avoid touching the inside wall and causing foaming as much as possible.
2. Decant the liquid from each well, do not wash. Immediately add 100μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37°C.
3. Decant the solution from each well，add 350μL of wash buffer to each well. Soak for 1-2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times. Note: a microplate washer can be used in this step and other wash steps. Make the tested strips in use immediately after the wash step. Do not allow wells to be dry
4. Add 100μL of HRP Conjugate working solution to each well. Cover with the Plate sealer. Incubate for 30 min at 37°C.
5. Decant the solution from each well, repeat the wash process for 5 times as conducted in step 3.
6. Add 90μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37°C. Protect the plate from light. Note: the reaction time can be shortened or extended according to the actual color change, but not more than 30min. Preheat the Microplate Reader for about 15 min before OD measurement.
7. Add 50μLof Stop Solution to each well. Note: adding the stop solution should be done in the same order as the substrate solution.
8. Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.