Neutralization Assay utilizing the HA-CoV-2 pseudovirus for titering antibody containing serum using multiple dilutions and 4-18hr incubation on a 96 well plate.
Purpose: Neutralization Assay utilizing the HA-CoV-2 pseudovirus for titering antibody containing serum using multiple dilutions and 4-18hr incubation on a 96 well plate.
Equipment, Supplies and Reagents
|Item||Source, Catalog #, Lot #|
|Human antibody (AB) serum||Patients or purchased AB solutions|
|Equipment Not Included|
|T75 Tissue Culture Flask||GenClone #25-209|
|Sterile Reagent Reservoir||Fisher #03-449-255|
|96-Well Cell Culture Plate||Genesee Scientific #25-109MP|
|96-Well Tissue Culture Plate, Greiner Bio-One (With Lid, µClear® White Flat Round, Chimney)||VWR #82050-758|
|GloMaxⓇ Discover Microplate Reader||Promega #GM3000|
|Reagents Not Included|
|DMEM (serum-free)||Thermofisher #11995-073|
|Fetal Bovine Serum (FBS)||Neuromics #FBS001|
|MEM Non-Essential Amino Acids Solution (100x) (NEAA)||Thermofisher #11140-050|
100units/mL(pen) + 100ug/mL(strep)
|Corning® Dulbecco’s Phosphate-Buffered Saline, 1X without calcium and magnesium (DPBS)||Corning #21-031-CM|
|TrypLE™ Express Enzyme (1X), phenol red (trypsin)||Thermofisher #12605-036|
|HEK293T(ACE2+TMPRSS2) cells, grown to confluence||Virongy Biosciences Inc. # RCSNAK-01|
|HA-CoV-2(Luc) pseudovirus||Virongy Biosciences Inc. # RCSNAK-01/RCSNAK-02|
|Standard SARS-CoV-2 Neutralizing Antibody||Virongy Biosciences Inc. # RCSNAK-01/RCSNAK-02|
|5X Cell Lysis Buffer||Virongy Biosciences Inc. # RCSNAK-01/RCSNAK-02|
|D-luciferin Solution||Virongy Biosciences Inc. # RCSNAK-01/RCSNAK-02|
|Luciferase Buffer Solution||Virongy Biosciences Inc. # RCSNAK-01/RCSNAK-02|
|1X Cell Lysis Buffer (10mL)
*All cell culture work needs to be done in a laminar airflow Class II biosafety cabinet and strict sterile procedures must be used.
- Initiate the HEK293T(ACE2+TMPRSS2) cell culture by thawing the frozen stock vial in a 37℃ water bath. Once thawed immediately transfer the tubes contents to 5 mL of prewarmed DMEM++.
- Centrifuge the cell for 5 minutes at 1200rpm and discard the supernatant. Resuspend the cells in 10mL of prewarmed DMEM++ and transfer the cells to a T75 flask
- Grow the cells to confluence before starting the Rapid SARS-CoV-2 Neutralizing Antibody Assay.
- Infection Procedure (24hrs Prior to Infection):
*All cell culture work needs to be done in a laminar airflow Class II biosafety cabinet and strict sterile procedures must be used. The HA-Cov-2(Luc) infection typically requires a minimum of 4-6 hours to complete. Start times must be considered beforehand.
- 24 hours prior to neutralization assay, remove confluent HEK293T(ACE2+TMPRSS2) cells from the flask by incubating the cells with 3mL TrypLE™ Express Enzyme (trypsin) for 5-10 minutes at 37℃.
- After the cells are detached from the flask, add 3mL DMEM++ medium and pipette vigorously to separate cells.
- Transfer the cells to a tube and count the cells.
- Prepare a 5mL solution of cells at 5x105cells/mL. Each well in a 96 well cell culture plate should receive 2.5x104 cells in 50uL of medium. Use a multi-channel pipettor and sterile basin to transfer 50uL of the cells into the respective wells. Note: exchange tips frequently to ensure consistent volumes.
- Incubate plate 12-18hrs (overnight) at 37℃.
- Plate Map Development
- Create a plate map for the 96-well plate before pipetting the AB serum and the VLPs. The sample plate map below incorporates 14 different serum samples and the controls. Note: This is a sample set up of the plate and will differ based on your experimental conditions.
Controls: Positive control antibody, Virus with medium alone, and cells only
- Infection Procedure (Day of Infection):
- In a sterile polypropylene 96-well plate or racked tubes, prepare the antibody(AB) serum and HA-CoV-2 mixtures (Infection Medium) according to the plate map.
- Add 8 µL of each sample AB to column 1 rows A-G and column 7 rows A-G. Add 8 µL of the positive control antibody to well H1. Note: Be sure to leave 3 wells for virus alone and 3 wells for a cell only control (H7-H12).
- Place 6 µL of serum-free DMEM in columns 2-6 and 8-12. Perform a serial dilution for each sample AB and the positive control AB according to the table below. Using a higher volume setting on the pipette, pipette-mix the AB serum and medium thoroughly before transferring to the next well. Note: Be sure to exchange pipette tips between well transfers and ensure that antibody serum and serum-free medium are mixed thoroughly to produce accurate results.
|Well #||Antibody Serial Dilutions||Particle Type (Volume)|
|8 µL of AB Serum||54 µL of HA-CoV-2(Luc)|
|2 µL of well #1 + 6 µL serum-free medium||54 µL of HA-CoV-2(Luc)|
|2 µL of well #2 + 6 µL serum-free medium||54 µL of HA-CoV-2(Luc)|
|2 µL of well #3 + 6 µL serum-free medium||54 µL of HA-CoV-2(Luc)|
|2 µL of well #4 + 6 µL serum-free medium||54 µL of HA-CoV-2(Luc)|
|2 µL of well #5 + 6 µL serum-free medium (After mixing discard 2uL)||54 µL of HA-CoV-2(Luc)|
Note: Remove 2 µL from the last AB dilution before adding HA-CoV-2(Luc) to maintain consistent volumes across wells.
- Add 54 µl of HA-CoV-2(Luc) to each AB containing well. Using a higher volume setting on a multi-channel pipette, pipette-mix the mixture.
- In wells H7-9 Prepare 3 negative control with 54 µL of HA-CoV-2(Luc) with 6 µL of serum free medium
- In wells H10-12 Prepare the cell only controls with 60 µL of serum free medium
- Seal the 96-well plate with plate sealer
- Incubate the HA-CoV-2(Luc) and AB mixture for 1hr at 37℃.
- After the 1hour incubation, apply 50 µL of Infection Medium and controls to the plated HEK293T(ACE2+TMPRSS2).
- Incubate the cells and infection medium at 37℃ for a minimum of 4 hours to a maximum of 18hrs post-infection before running the luciferase assay according to the protocol below. (For best results allow the infection to run overnight)
Antibody + Virus Complex
+ 50 µL DMEM++ media
|A||1 : 20|
|B||1 : 80|
|C||1 : 320|
|D||1 : 1280|
|E||1 : 5120|
|F||1 : 20480|
- Luciferase Assay Protocol
- Following the incubation with the Infection Medium remove cell supernatant completely and add 100ul of 1X Cell Lysis Buffer directly over the cells to ensure complete lysis. Keep the 96-well plate on ice after this step. Pipette up and down to ensure complete mixing. Allow cells to lyse for at least 5 minutes.
- In a dark location, away from direct light, prepare the Firefly Luciferase Assay Solution by mixing the D-Luciferin Solution with the Substrate Buffer in a 1:50 ratio. For a full 96 well plate prepare 5.5 mL of the luciferase substrate solution by mixing 110 µL of the D-Luciferin Solution with 5.390 mL of the Substrate Buffer. Note: Use the Firefly Luciferase Assay Solution within 30 minutes of preparation. Do not reuse the Firefly Luciferase Assay Solution.
- Add 50 µL of the Firefly Luciferase Assay Solution into a white 96-well microplate.
- Apply 50 µL of the cell lysate from each sample to Firefly Luciferase Assay Solution loaded plate.
The plate should be run within 10-15 minutes of adding the samples to the plate. (Tip: Run the plate reader in a dark room to reduce any background signal.) Note: More lysate can be added to generate a stronger luciferase signal.