SARS-CoV-2 Variant Inhibitor Screening Kit

Assay Procedure

Sample Assay Protocol

Assay Procedure

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.

1. 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.)
2. Cover with an ELISA plate sealer and incubate overnight at 2-8 °C.
3. 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.
4. Add 200 µL/well Blocking Buffer and incubate for 60-90 minutes at 37 °C.
5. 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.
6. 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.)
7. Incubate for 60-90 minutes at room temperature.
8. In the Sample Dilution Plate (clear 96-well microplate) add 60 μL/well Assay Buffer to all wells of the plate.
9.  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).
10. 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.
11. 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.
12. Incubate for 60-90 minutes at room temperature.
13. 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.
14. Gently tap the plate to ensure thorough mixing. Incubate for 90 minutes at room   temperature.
15. 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.
16.  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.)
17. Incubate for 30 minutes at room temperature.
18. 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.
19. Add 100 µL of Substrate Solution to each well. Protect from light.
20. Incubate for 15-25 minutes at room temperature.
21. 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.
22. Determine optical density (OD) of each well within 15 minutes at 450 nm with a correction wavelength at 540 or 570 nm.
23. Calculation: If samples are tested in duplicates, calculate average OD. Use Average OD to calculate % Blocking: