SARS-CoV-2 Spike Protein Serological IgG ELISA Protocol
This ELISA Kit Is Intended For Research Use Only. Not For Use in Diagnostic or Clinical Procedures.
A. Solutions and Reagents
NOTE: Prepare solutions with deionized/purified water or equivalent. Prepare only as much reagent as needed on the day of the experiment.
- Spike Protein Coated Microwells: Bring all to room temperature before opening bag/use. Unused microwell strips should be returned to the original re-sealable bag containing the desiccant pack and stored at 4°C.
- 1X ELISA Wash Buffer: Prepare by diluting ELISA Wash Buffer (20X) (included in each kit) to 1X with deionized water.
- Sample Diluent A: Diluent provided for dilution of samples and for reconstitution of Positive and Negative Controls included in kit.
- HRP Diluent: Red colored diluent for reconstitution and dilution of the Anti-Human IgG, HRP-linked Antibody (11 mL provided).
- Anti-Human IgG, HRP-linked Antibody (ELISA Formulated): Supplied lyophilized as a red colored cake or powder. Add 1.0 mL of HRP Diluent (red solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. To make the final working solution, add the full 1.0 mL volume of reconstituted HRP-linked Antibody to 10.0 mL of HRP Diluent in a clean tube and gently mix. For best results, use this working solution immediately. Unused working solution may be stored for up to 4 weeks at 4°C, although there may be some loss of signal compared to freshly made solution.
- Positive Control: Reconstitute the vial of lyophilized Positive Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Positive Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Positive Control signal if freeze/thawed). Positive Controls are supplied as a control reagent, not as an absolute quantitation measure.
- Negative Control: Reconstitute the vial of lyophilized Negative Control with 1.0 mL Sample Diluent A. Mix thoroughly and gently, hold at room temperature for 1 minute and then follow the steps outlined below in the “Test Procedure” section. Negative Controls are recommended to be used immediately after reconstituting, however remaining material may be stored at -80°C (there may be some loss of the Negative Control signal if freeze/thawed).
- TMB Substrate (#7004): Bring to room temperature before use.
- STOP Solution (#7002): Bring to room temperature before use.
B. Test Procedure
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
- Prepare all reagents as indicated above (Section A).
- Human-sourced samples should be handled in accordance with accepted safety practices. Samples should be diluted at least 1:800 with Sample Diluent A and can be further serially diluted if relative quantification is needed by the user. Positive and Negative Controls do NOT need to be diluted after reconstitution. Refer to the datasheet which shows typical results observed for the Positive Control, Negative Control, serum from uninfected individuals, and serum from SARS-CoV-2 patients. When using the cutoff criteria described below to determine if a sample is positive for anti-CoV-2 Spike Protein antibodies, samples diluted 1:800 must be compared to the undiluted Negative Control.
NOTE: Sample storage/handling, including heat-inactivation of samples, can potentially affect observed signals. Therefore, it is strongly recommended that in addition to the Positive and Negative Controls included with the kit, the user includes their own negative and positive patient samples as controls when running the assay in order to establish an appropriate cutoff value.
- Add 100 μL of each diluted sample, Positive Control, Negative Control, and blank (Sample Diluent A only) to the appropriate wells. Seal the plate with the supplied sealing tape and incubate for 1 hour at 37°C.
- Gently remove the tape and wash wells:
- Discard plate contents into a receptacle.
- Wash 4 times with 1X ELISA Wash Buffer, 200 μL each time for every well. After each wash, aspirate or decant from wells. Invert the plate and blot it against clean paper towels to remove the residual solution in each well, but do not allow wells to completely dry at any time.
- Clean the underside of all wells with a lint-free tissue.
- Add 100 μL of reconstituted Anti-Human IgG, HRP-linked Antibody (ELISA Formulated). Seal with tape and incubate the plate for 30 min at 37°C.
- Repeat wash procedure (Section B, Step 4).
- Add 100 μL of TMB Substrate to each well. Seal with tape and incubate the plate in the dark for 10 min at 37°C.
- Add 100 μL of STOP Solution to each well. Shake gently for a few seconds.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
- Read results:
- Visual Determination: Read within 30 min after adding STOP Solution.
- Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP Solution.
- Data Analysis:
- Subtract “blank” well (Sample Diluent A only) absorbance 450 nm values from sample, Positive, and Negative Control values.
- Positive Control Values should be > 1.5 and Negative Control Values should be < 0.75.
- Samples (1:800 dilution) are considered positive if they are greater than 4.1 x Negative Control absorbance 450 nm value.
- Samples (1:800 dilution) are considered negative if they are less than 3 x Negative Control absorbance 450 nm value.
- Samples (1:800) are considered inconclusive if they are greater than the 3 x Negative Control absorbance 450 nm value and less than 4.1 x Negative Control absorbance 450 nm value.
- Limitations: Experimental cutoffs were determined by assaying a set of confirmed SARS-CoV-2 positive samples and uninfected donor serum collected prior to the SARS-CoV-2 pandemic. Researchers can establish or modify this cutoff using additional samples. Positive or negative results from this assay should not be the sole basis for determining the infection status of a sample. A negative result can occur in SARS-CoV-2 patient samples due to:
- improper sample handling/storage
- timing of sample collection post-infection
- patients having impaired immune function
Protocol Id: 2024