COVID-19 (IgM) ELISA Kit

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.

This ELISA kit uses Indirect-ELISA as the method to qualitatively detect the SARS-CoV-2 Nucleocapsid protein IgM in the sample. The micro ELISA plate provided in this kit is pre-coated with purified SARSCoV-2 Nucleocapsid protein antigen, after adding samples to wells, the SARS-CoV-2 Nucleocapsid protein IgM in the samples will combine with the pre-coated SARS-CoV-2 Nucleocapsid protein antigen.

After washing completely, add Horseradish Peroxidase (HRP) conjugated mouse anti human IgM to develop the antigen-antibody-HRP conjugated secondary antibody complex. Free components are washed away, then the substrate solution is added to each well. Only those wells that contain SARS-CoV2 Nucleocapsid protein IgM and HRP conjugated anti-human IgM will appear blue in color. The enzymesubstrate 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. Compared with the CUT OFF value to judge whether SARS-CoV-2 Nucleocapsid protein IgM exists in the tested samples or not.

• 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

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.

Before test, prepare the samples as the following instructions.

Sample dilution: Dilute the tested serum or plasma at 100 fold by using the Sample & Control Diluent, mix thoroughly.

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.

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.Positive Control and Negative Control working solution: Centrifuge the Positive Control or
Negative Control at 10,000×g for 1 min. Add 0.5 mL of Sample & Control Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette.

3.Concentrated HRP Conjugated Mouse anti-human IgM working solution: Calculate the required amount before the experiment (100μL/well). In preparation, slightly more than calculated should be prepared. Dilute the 100×Concentrated HRP Conjugated Mouse anti-human IgM to 1× working solution with HRP Conjugate Diluent.

4.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.

1.Determine wells for Positive Control, Negative Control, Blank (Do not add any reagents except Substrate Reagent and Stop Solution) and Samples. Add 100μL of controls and samples to the appropriate wells (It is recommended that all controls and samples be assayed in duplicate.). Cover the plate with the sealer provided in the kit. Incubate for 45 min at 37„ƒ.

2.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.

3.Add 100μL of HRP Conjugated Mouse anti-human IgM working solution to each well (except the blank well). Cover with the Plate sealer. Incubate for 30 min at 37°C.

4.Decant the solution from each well, repeat the wash process for 5 times as conducted in step 2.

5.Add 90μL of Substrate Reagent to each well (including the blank 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. Pre-heat the Microplate Reader for about 15 min before OD measurement.

6. Add 50μL of Stop Solution to each well (including the blank well). Note: adding the stop solution should be done in the same order as the substrate solution.

7.Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.