Protocol

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 #
Samples
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
Penicillin/Streptomycin (Pen/Strep)

100units/mL(pen) + 100ug/mL(strep)

LifeTech #15140-122
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
Reagents Included
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
Reagent Preparation
DMEM++ (500mL)

  1. 450 mL DMEM Serum Free
  2. 50 mL FBS, heat activated and filtered so it is sterilized
  3. 5 mL NEAA
  4. 1µg/mL Puromycin (Selection Agent for ACE2/TMPRSS2 Expression)
  5. 4µg/mL Hygromycin B (Selection Agent for ACE2/TMPRSS2 Expression)
  6. 100units/mL Pen/Strep
1X Cell Lysis Buffer  (10mL)

  1. 8mL PBS
  2. 2mL 5X Cell Lysis Buffer

 

Before Starting: 

*All cell culture work needs to be done in a laminar airflow Class II biosafety cabinet and strict sterile procedures must be used.

  1. 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++.
  2. 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
  3. Grow the cells to confluence before starting the Rapid SARS-CoV-2 Neutralizing Antibody Assay.

 

  1. 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.

  1. 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℃.
  2. After the cells are detached from the flask, add 3mL DMEM++ medium and pipette vigorously to separate cells.
  3. Transfer the cells to a tube and count the cells.
  4. 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.
  5. Incubate plate 12-18hrs (overnight) at 37℃.
  1. Plate Map Development
  1. 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

 

  1. Infection Procedure (Day of Infection):
  1. 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.
  1. 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).
  2. 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)
1A 

(1:10) AB

8 µL of AB Serum 54 µL of HA-CoV-2(Luc)
2A

(1:40) AB

2 µL of well #1 + 6 µL serum-free medium 54 µL of HA-CoV-2(Luc)
3A

(1:160) AB

2 µL of well #2 + 6 µL serum-free medium 54 µL of HA-CoV-2(Luc)
4A

(1:640) AB

2 µL of well #3 + 6 µL serum-free medium 54 µL of HA-CoV-2(Luc)
5A

(1:2560) AB

2 µL of well #4 + 6 µL serum-free medium 54 µL of HA-CoV-2(Luc)
6A

(1:10240) AB

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.

  1. 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.
  2. In wells H7-9 Prepare 3 negative control with 54 µL of HA-CoV-2(Luc) with 6 µL of serum free medium
  3. In wells H10-12 Prepare the cell only controls with 60 µL of serum free medium
  4. Seal the 96-well plate with plate sealer

 

  1. Incubate the HA-CoV-2(Luc)  and AB mixture for 1hr at 37℃.
  2.  After the 1hour incubation, apply 50 µL of Infection Medium and controls to the plated HEK293T(ACE2+TMPRSS2).
  3. 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)

 

Final Dilutions

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

 

  1. Luciferase Assay Protocol
  1. 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.
  2.  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.
  3. Add 50 µL of the Firefly Luciferase Assay Solution into a white 96-well microplate.
  4. 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.