SARS-CoV-2 Variant Inhibitor Screening Kit New
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SARS-CoV-2 Variant Inhibitor Screening Kit Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This SARS-CoV-2 Variant Inhibitor Screening Kit contains the basic components required to measure the ability of the sera of vaccinated donors to block the binding between RBDs of COVID-19 variants and human ACE-2. The suggested diluent is suitable for the analysis of most serum and plasma samples.
See the Data: Read our white paper on testing individual serum samples using this kit. Download White Paper
Assay Workflow: 5 Simple Steps
- Quickly and efficiently evaluates the effectiveness of vaccines against SARS-CoV-2 variants
- Validated step by step assay protocol
- No special equipment needed
- Can be used for high throughput screening SARS-CoV-2 inhibitors
Background: Severe acute respiratory syndrome Coronavirus-2
Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, has posed a serious threat to global human health. With the emergence of variants, especially the Variants of Concern (VOCs), a quick and effective way to assess how effective the current vaccines are against the major variants is needed. The COVID-19 Neutralization Kit is an ELISA type of binding assay measuring the ability of the sera of vaccinated donors to block the binding between RBDs of COVID-19 variants and human ACE-2.
- Capture Antibody
- SARS-Cov-2 Spike RBD Wild Type
- SARS-CoV-2 Spike RBD Variants (Alpha, beta, Gamma and Delta)
- Biotinylated Human ACE-2
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
Other Reagents Required
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Figure 1: Example data using vaccinated donor samples. Serological samples were collected from two donors over a time course consisting Pre-Vaccination (Pre-Vac), or after receiving the 1st Dose or 2nd Dose of the Moderna Vaccine. Donor 1 (A-C) and Donor 2 (D-F) had different blocking antibody levels after the first and second doses. In addition, increasing amounts of blocking antibodies were present at higher samples Dilution Factors after each dose. The differences in blocking efficiency of ACE-2 association between the WT, Alpha, Beta, Gamma, or Delta variant Spike RBD was measured.
Preparation and Storage
Background: Spike RBD
The coronavirus Spike protein receptor binding domain (RBD) resides within the S1 subunit and is responsible for binding to host cell receptors and initiating viral infection. Past coronaviruses SARS and MERS along with the global pandemic caused by SARS-CoV-2 have sparked great interest and scientific discovery leading to new vaccines and drug development. At the heart of coronavirus biology lies the Spike protein and its receptor binding domain. The Spike RBD is a 26 kDa domain consisting of a twisted five-stranded antiparallel beta sheet and sits at the apex of each Spike protein monomer. The Spike RBD is flexible thanks to a hinge region that allows for conformational changes that expose (up or open conformation) or hide (down or closed conformation) its receptor contacts. For SARS-CoV-2, the Spike RBD recognizes and tightly binds to the human receptor ACE-2. In the closely related MERS coronavirus, human DPPIV/CD26 acts as the receptor.
Sample Assay Protocol
Prepare a Plate Layout listing how to coat the desired RBD protein(s) to the Assay Plate and which wells to add each sample. It is suggested to dilute samples in the sample Dilution Plate and then transfer to the Assay Plate. Plate layouts for the Dilution Plate and Assay Plate are identical. Note:Store Biotinylated Human ACE-2 (1000X) at 2-8 °C. Bring the remaining buffers and plates to room temperature for a minimum of 15 minutes before use. Do not stack plate for any part of the procedure.
- Dilute His-Tag Capture Antibody from 200X to 1X with ELISA Coating Buffer/PBS. Coat the His-Tag Capture Antibody on a 96 well Strip-well Microplates (R&D Systems®, Catalog # DY008) or (R&D Systems, Catalog # DY990), 100 µL/well. This is the Assay Plate. (e.g., for every 12 mL of 1X Capture Antibody needed, add 60 µL of His-Tag Capture Antibody (200X) and 11.94 mL of ELISA Coating Buffer/PBS.)
- Cover with an ELISA plate sealer and incubate overnight at 2-8 °C.
- The next day, aspirate each well and wash 4 times with 1X Wash Buffer (360 µL/well). Rotate the plate 180 degrees after the second wash. After the last wash, invert plate and blot on clean paper towels.
- Add 200 µL/well Blocking Buffer and incubate for 60-90 minutes at 37 °C.
- Following the incubation, remove the Assay Plate from 37 °C and place at room temperature for a minimum of 15 minutes. Aspirate each well and invert plate and blot on clean paper towels.
- Refer to the Plate Layout to coat the desired RBD protein(s) to the Assay Plate. Prepare a dilution of the desired SARS2 RBD protein from 1000X to 1X in Assay Buffer and add to the plate, 100 µL/well. (e.g., for every 12 mL of 1X SARS2 RBD protein needed, add 12 µL of SARS2 RBD protein from (1000X) and 11.99 mL of Assay Buffer.)
- Incubate for 60-90 minutes at room temperature.
- In the Sample Dilution Plate (clear 96-well microplate) add 60 μL/well Assay Buffer to all wells of the plate.
- Refer to the Plate Layout for which wells to add each sample. Add 30 µL of testing sample to the first wells (For serum or plasma samples, we suggest a 1:9 pre-dilution with Assay Buffer). Mix 30 µL/well and make 3-fold serial dilutions, discard the remaining 30 µL/well from the last wells. Do not add testing sample to RBD only and Background control wells. (e.g., for every 324 µL 1:9 pre-diluted sample needed (or enough to use in duplicate for five RBD proteins), add 36 µL serum or plasma samples to 288 µL of Assay Buffer to produce a f inal volume of 324 µL 1:9 pre-diluted sample).
- Aspirate each well of the Assay Plate and wash 4 times with 1X Wash Buffer (360 µL/well). Rotate the plate 180 degrees after the second wash. After the last wash, invert plate and blot on clean paper towels.
- Mix and transfer 50 µL/well from the Sample Dilution Plate to the Assay Plate. Use new tips for each well. After transferring, discard the Working Plate.
- Incubate for 60-90 minutes at room temperature.
- Prepare a dilution of Biotinylated Human ACE-2 from 1000X to 1X in Assay Buffer and add 50 µL/well to all wells of the Assay Plate making the final volume 100 µL/well. Use new tips for each well. (e.g., for every 12 mL of 1X Biotinylated Human ACE-2 needed, add 12 µL of Biotinylated Human ACE-2 (1000X) and 11.99 mL of Assay Buffer.) Note: Keep Biotinylated Human ACE-2 (1000X) on ice while preparing dilutions.
- Gently tap the plate to ensure thorough mixing. Incubate for 90 minutes at room temperature.
- Aspirate each well and wash 4 times with 1X Wash Buffer (360 µL/well). Rotate the plate 180 degrees after the second wash. After the last wash, invert plate and blot on clean paper towels.
- Prepare a dilution of Streptavidin-HRP Conjugate at a 1:200 dilution in Assay Buffer and add 100 µL/well to all wells. (e.g., for every 12 mL of 1:200 Streptavidin-HRP Conjugate needed, add 60 µL of SA-HRP Conjugate and 11.94 mL of Assay Buffer.)
- Incubate for 30 minutes at room temperature.
- Aspirate each well and wash 4 times with Wash Buffer (1X) (360 µL/well). Rotate the plate 180 degrees after the second wash. After the last wash, invert plate and blot on clean paper towels.
- Add 100 µL of Substrate Solution to each well. Protect from light.
- Incubate for 15-25 minutes at room temperature.
- Add 50 µL of Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
- Determine optical density (OD) of each well within 15 minutes at 450 nm with a correction wavelength at 540 or 570 nm.
- Calculation: If samples are tested in duplicates, calculate average OD. Use Average OD to calculate % Blocking:
What can cause high background?
Problem of high background may be due to insufficient washing or extended incubation time with detection reagents, among other factors.
What are possible reasons for low assay signal?
Low assay signal may be due to non-optimal preparation of Recombinant SARS2 Spike RBD proteins or Biotinylated Human ACE-2. Be sure to review the product insert and follow reconstitution and dilution instructions for each lyophilized reagent. Low signal may also be caused by insufficient incubation time for color reaction. Color development for the Covid-19 Neutralization assay requires 15 to 25 minutes.
What could cause high variability between sample duplicates?
The two main reasons for high variability in this assay are non-optimal pipetting technique and samples not well mixed. Ensure a consistent and accurate pipetting method. Mix sample thoroughly and dispense accurately. Change pipette tips between samples. Ensure that your pipettes are calibrated.
Is it okay to modify the plate layout in the kit product datasheet?
Yes, a plate layout is provided in the kit insert as an example, and investigators can modify the plate layout to suit specific needs. Spike RBD WT or variant proteins are supplied at higher amounts to provide flexibility for investigators to use each protein of interest as desired.
Why are Sample Dilution Plates provided in the kit?
The Sample Dilution Plates have reduced protein binding to minimize loss of sample during the dilution and pre-incubation steps, and the plates are provided in the kit for convenience of use. Diluted samples prepared in a dilution plate can be transferred to a 96-well Assay Plate in an efficient manner minimizing chance for assay drift.
The SARS-CoV-2 variants that I am interested in are not listed in the kit insert. Is there an option to obtain a kit that contains other variants?
Yes; to discuss other available options, please contact our Technical Support by email email@example.com or by phone Toll Free USA, Canada: 1-800-343-7475
Phone (International): 1-800-343-7475.
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