Document text
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 1
TABLE OF CONTENTS
1.HISTORICAL REVISION SUMMARY ............................................................................... 2
Table 1. Chronological Summary of Previous Versions ........................................................ 2
2.PURPOSE ............................................................................................................................. 2
3.SCOPE ................................................................................................................................... 2
4.GLOSSARY ........................................................................................................................... 2
Table 2. Terms and Definitions .................................................................................................. 2
5.GENERAL ............................................................................................................................. 3
6.RESPONSABILITIES .......................................................................................................... 4
Table 3. Roles and Responsibilities .......................................................................................... 4
7.SAFETY ................................................................................................................................ . 5
8.MATERIALS AND EQUIPMENT ....................................................................................... 5
9.PROCEDURE ....................................................................................................................... 6
9.1.Day 0: Preparation of ROPs ............................................................................................... 6
9.1.1. Seeding Cells ................................................................................................................. 6
9.2.Day 1: Preparation of SAPs ................................................................................................ 8
9.2.1. Sera Dilution/Infection ................................................................................................... 9
9.2.2. Processing of SAPs .................................................................................................... 10
9.2.3. Preparation of Viral Stock Solution ........................................................................... 11
9.2.4. Addition of Viral Stock Solution to SAPs and Neutralization of Virus .................. 11
9.2.5. Transfer Neutralization Reaction onto ROPs and Decontamination of SAPs ....11
9.3.Day 2 : Acquire images ...................................................................................................... 12
9.3.1. Preparation and Addition of Hoechst 33342 Solution to ROPs ............................ 13
9.3.2. Image Acquisition and Counting on the Cytation 7 ................................................ 13
9.4.Analysis of Data and Calculation of SARS -CoV-2_NT Titers ..................................... 13
9.5.SARS -CoV-2_NT Data and Document Review ............................................................ 13
10.REFERENCES ................................................................................................................... 14
Table 6. Form References ........................................................................................................ 14
11.DOCU MENT VERSION MODIFICATIONS ................................................................... 14
Table 7. Detailed Changes ...................................................................................................... 14
FDA-CBER-2021-5683-0142291
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 2
Document Approval Record ..................................................................................................... 15
1.HISTORICAL REVISION SUMMA RY
Table 1. Chronological Summary of Previous Versions
Version # Effective Date Originator Summary
2.0 Current Pei-Yong Shi Approved.
1.1 2020 0614 Xuping Xie Revised…
1.0 20200529 Camila Fontes New Document
2.PURPOSE
This document describes the procedure for a manual 96 -well SARS -CoV-2-mNG
reporter virus neutralization assay (SARS -CoV-2_NT) designed to measure functional,
neutralizing antibodies specific for SARS -CoV-2 in test serum using Cytation 5 Image
Reader.
3.SCOPE
The scope applies to all personnel in the Shi Lab at UTMB responsible to support Pfizer
clinical study.
NOTE: Procedures for other UTMB and Pfizer groups may vary in format, content,
numberi ng, and approval procedures.
4.GLOSSA RY
Table 2 . Terms and Definitions
TERM DEFINITION
Ab Antibody
Batch A sample list for an assay. One batch contains all samples tested in
a single assay run.
BSC Biological Safety Cabinet
Cytation 7 Cell imaging multi -mode reader manufactured by Biotek
FBS Fetal Bovine Serum
FFU Focus Forming Units
Gen5 Software associated with the Cytation 7
FDA-CBER-2021-5683-0142292
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 3
GMT Geometric Mean Titer
HBSS Hanks' Balanced Salt Solution
Hoechst 33342 Cell-permeant nuclear counterstain that emits blue fluorescence
when bound to dsDNA
Monolayer Cell culture in which cells are grown in a single homogenous layer
PPE Personal Protective Equipment
PBS Phosphate Buffered Saline
ROP Read Out Plate
SARS -CoV-
2_NTManual 96 -well neutralization assay for the detection of functional
antibodies to SARS -CoV-2
SARS -CoV-2
Critical Reagent
Reference FormSARS -CoV-2_NT reference document that contains clinical or
developmental study -specific information, eg, viral bank number,
viral bank date, viral st ock solution dilution, and staining antibody
information. One critical reagent form is created prior to the start of
each clinical study or assay development activity.
QCS Quality Control Sample
QNS Quantity Not Sufficient
SAP Sample Assay Plate
SARS -CoV-2 Severe acute respiratory syndrome coronavirus 2
SARS -CoV-2-
mNGSARS -CoV-2-mNeonGreen reporter virus
SOP Standard Operating Procedure
Titer The reciprocal of the dilution of a test serum required to neutralize
virus at a specified percentage of the viral input in a neutralization
assay
VSS Viral Stock Solution
Vero African green monkey kidney epithelial cells
5.GENERA L
The SARS -CoV-2_NT is a biofunctional assay designed to measure total serum
antibody levels capable of neutralizing SARS -CoV-2 infection of cells in culture.
The SARS -CoV-2-NT is a three -day assay, performed manually. On Day 0, African
green monkey kidney epithelial cells (Vero) are seeded into 96 -well tissue -culture
treated plates. On Day 1, serial dilutions of heat -inactivated i mmune sera in sample
assay plates (SAPs) are incubated with SARS -CoV-2-mNG reporter virus for 60
minutes to allow antigen specific antibodies to bind to the virus. The reaction is then
transferred onto the Vero cell monolayer read -out plates (ROPs) and allowed to
incubate overnight; only non -neutralized virus is able to infect the cells. On Day 2,
productive viral infection is detected in live cells using the mNeonGreen expressed by
FDA-CBER-2021-5683-0142293
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 4
the reporter virus. Live cells are stained with Hoechst 33342. Both vir al foci and live
cells are enumerated from the ROPs on a Cytation 7 reader.
A sample titer is calculated as the reciprocal serum dilution at which a specific
percentage of the virus is neutralized (eg, 50% or 90% of the virus). This value is
referred to as the titer determining value (TDV). To calculate this percentage, an
infection rate must first be determined by dividing the number of viral foci by live cells in
any given well. The percent neutralization is then determined by comparing the
infection rates of the serum containing wells to that of the control wells without serum
samples. A titer is calculated for each of the two replicates of a sample and the
geometric mean titer (GMT) of the two is reported as the final sample titer.
The initial serum dilution for the SARS -CoV-2_NT is 1:20, which, along with a 9 -point 2 -
fold serum dilution series, enables a potential plate titer range of 20 to 5,120. Samples
that receive titers greater than 5,120 are retested after pre -dilution in assay buffer.
Samp les that fail to neutralize at the TDV when tested at the lowest serum dilution of
1:20 are reported to have a neutralizing titer of 10, one half of the LOD of 20.
A quality control sample (QCS) is run on each assay plate to assess plate and run
suitabili ty. Plating of Vero onto the ROPs occurs at least 20 hours prior to the infection
of the monolayer. This incubation time allows the cells to adhere to the plate surface
and grow to a confluent monolayer to allow for consistent measurement of infection.
6.RESPONSA BILITIES
All personnel must betrained and knowledgeable with the process. Personnel can
have multiple roles within the process but may only sign once with the most
appropriate approval code.
Table 3 . Roles and Responsibilities
Roles Responsibilities
SARS -CoV-2_NT
-Analyst
-Coordinator
-Data Analyst
CFRead, understand, and complete training and qualification on
this test method.
Read, understand, and complete training on applicable
equipment operating manuals, procedures and test methods
that are required to execute the procedure described in this
document.
Execute this test method and complete SHI-TM-10011 -FM01
and SHI-TM-10011-FM02.
Consult with the PI if there are any questions related to the
procedure itself or serum samples, i f an atypical event is
FDA-CBER-2021-5683-0142294
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 5
observed or if a protocol deviation occurs.
Provides detailed scheduling of all SARS -CoV-2_NT runs.
Creates assay run batches and corrects run information with
QNS information from the annotated SHI -TM-10011 -FM01 .
Prepare SARS -CoV-2_NT assay plates
Maintain and grow Vero cells.
Plate the appropriate concentration of Vero cells
Reviewer XP Review SARS -CoV-2_NT documentation to ensure
completeness of data entry and compliance with Shi Lab
policies. Direct the SARS -CoV-2_NT Analyst to correct
documentation errors.
Verify that documentation is complete and compliant.
Supervisor PY Read and understand this test method.
Review and approve this test method.
Ensure that SARS -CoV-2_NT analysts executing this test
method are trained in the appropriate procedures and test
methods.
Provide guidance to SARS -CoV-2_NT analysts and reviewers
and answer any questions that arise during the execution of this
procedure.
7.SAFETY
Biosafety level 3 (BSL -3) precautions are required for any work with SARS -CoV-2
virus and has to be handled in accordance with BSL -3 procedures.
oPrior to starting work at BSL3:
oRegister as BSL3 worker and complete the training and access requirements
for BSL3 facilities:
UTMB BSL3 training course
BSL3 facility specific training and access checklist form
BSL3 check off
BSL3 mentorship
All laboratory work is to be carried out in compliance with UTMB safety policies and
generally accepted laboratory safety guidelines.
Refer to Laboratory Safety Reference Manual found on UTMB Website at
https://liveutmb.sharepoint.com/:b:/r/sites/collaboration/webfiles/Shared%20Docume
nts/EHS/ Website_Files/UTMB%20Safety%20Manual%202019_3 -5-
20.pdf?csf=1&web=1&e=lKehru
8.MATERI ALS AND EQUIPMENT
FDA-CBER-2021-5683-0142295
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 6
Cell bank of Vero African green monkey kidney epithelial cells (ATCC CCL81). Each
vial is used from p1 to p35 .
Viral stock of SARS -CoV-2 mNeonGreen, prepared in individual use aliquots (virus
construction described in Xie et al 2020). Viral stock is at ~2x107PFU/mL.
Black µCLEAR flat -bottom 96 -well plates (Greiner Bio -one™ 655090)
Corning™ Clear Polystyrene 96 -Well Microplates, round bottom (Corning Cat. No:
3799)
DMEM (Gibco Cat. No: 11965 -092)
Fetal Bovine Serum (Hyclone Cat. No.: SH30071.03)
Penicillium -Streptomyc in (P/S) (10,000 U/ml) (Gibco Cat. No: 15140 -122)
Gibco™ Trysin -EDTA (0.25%), phenol red (Gibco Cat. No.: 25200072)
Phosphate Buffered Saline (PBS) solution, pH 7.4 (Gibco Cat. No.: 10010 -023)
Gilson™ Accessory for PIPETMAN Complete Pipetting System
Reage nt Reservoirs 25 ml (Gilson Cat. No.: F267660) & 50 ml (Gilson Cat. No.:
F267670)
Cell Counting slides for TC10™/TC20™ Cell Counter, dual -chamber (Bio -Rad Cat.
No.: 1450011)
Corning™ Cell Culture Treated flasks .2µM Vent Cap T175cm2 (Corning™ Cat. No.:
431080)
VACUBOY Hand Operator (INTEGRA Biosciences)
Eppendorf Xplorer pipette, electronic 12 channel pipette 15 -300 µL (Eppendorf Cat.
No.: 4861000031)
TC20™ Automated Cell Counter (Bio -Rad)
Dualfilter 200ul (filtered tips) (Eppendorf Cat. No.:0030077.555)
Hank’s Balanced Salt Solution (HBSS) (1X) (Gibco Cat. No:14025 -092)
Breath -Easy sealing membrane. (Diversified Biotech Cat No.: BEM -1)
Tissue culture CO 2incubator
Cell Imager or inverted microscope
Microplate Reader (BioTek) (Cytation 7 Cell Imaging Multi -Mode Reader)
Hoechst 33342 (20mM) (Thermo Scientific Prod # 62249)
DMEM (1X) (Phenol Red Free) (Gibco Cat. No: 31053 -028)
GlutaMAXTM -1 (100X) (Gibco Cat. No: 35050 -061)
9.PROCEDURE
9.1. Day 0: Preparation of ROPs
oNote: Refer to Tables 3 -9 of SHI-TM-10011-FM01.
oEnsure the incubators are reading at 37°C/5% CO 2. Confirm that water
reservoir is full.
9.1.1. Seeding Cells
oSeed cells into 96 -well plate Black Microplates.
FDA-CBER-2021-5683-0142296
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 7
oRemove the cotton filter from 2ml serological pipette and assembly on the
hand operator of the VACUBOY
oRemove the medium from the cells using the VACUBOY.
oBriefly rinse the cell layer with 25ml PBS (same volume as the growth
medium) to remove all traces of serum that contains trypsin inhibitor. Remove
PBS using the VACUBOY. Repeat, wash twice.
oNote: make sure rinse every corner of the flask to get rid of any trypsin
inhibitor.
oAdd 3ml Trypsin -EDTA solution. Observe cells under an inverted microscope
until cell layer is dispersed (usually within 2 minutes at RT).
oNote: The activity of trypsin decreases when store dat 4°C. Do not store
trypsin at 4°Cfor > 1 week.
oHit/tap the flask to detach cells, add 10 mL of complete growth medium and
shake the flask to let the medium cover all the trypsinazed cells in order to
inhibit the trypsin. Aspirate the cell susp ension by gently pipetting and
transfer the cell suspension into 50ml falcon tube.
oCentrifuge, RT, 3min, 1200rpm (300g)
oRemove media carefully don’t get too close to the cells.
oResuspend cells in phenol red free media (DMEM phenol red free, 2% FBS,
1% P/S, 2% GlutaMAX). Disperse cells by tapping the tube with finger gently,
this is critical steps make sure cells are evenly dispersed. Add 5ml of media,
keep tapping and swirling to resuspend, add more media (10 -20 ml mix by
pipetting up/down). Note: Using phenol red free media reduces the
background when reading the plate.
oCount using the cell counter (C -Chip DHC -N01-5). Count live cells. Get a
microcentrifuge tube and mix 50ul of trypan blue with 50ul of cell suspension.
Add mixture to both sides of count ing slide, count both sides to get the
average of live cells.
oDilute to 2.4×105 cells/ml. Add 50 µl of the diluted cell suspension to each
well in the 96 -well plate (ROP) to reach 1.2×104 cells/well. Note: to make
sure the cells are evenly distributed in the 96 -well plate. Leave the plate on
the bench after seeding about 5 -10min, then hit/tap the plate gently to make
cells evenly distribute in the wells.
Example: 1 -96W plate 50ul/well, live cell count= 1.0×106
FDA-CBER-2021-5683-0142297
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 8
oAdd 1 -5 ml of the remaining cell suspension to new culture flask to continue
to grow cells depending on your needs.
oIncubate cell plates at 37°C. 6. Ensure ROPs have incubated at least 20
hours prior to execution of Transfer of Neutralization Reaction onto ROPs
9.2. Day 1: Preparation of SA Ps
oNote: Refer to Tables 3 -9 of SHI -TM-10011-FM01.
oEnsure the incubators are reading at 37°C/5% CO 2. Confirm that water
reservoir is full.
oInspect 6 wells at r andom of 2 ROPs per run under the light microscope to
ensure the monolayer is healthy with no microbial contamination and has
≥80% confluency. A healthy Vero monolayer is evenly dispersed squamous
cells adhered to the bottom of the ROP well. Record abnormalities in Table
13 of SHI -TM-10011 -FM01.
oROP . Label and number each plate appropriately (typically 1 -12) in the
Biosafety hood with marker and store in 37 °C/5% CO 2until transfer.
ROP lid labeling
FDA-CBER-2021-5683-0142298
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 9
ROP side labeling
oHeat inactive all the sera at 56C, 30mins (If needed)
9.2.1. Sera Dilution/Infection
oSAP. Take a new 96 -well plate with lid (clear plate, for dilutions). Label and
number each plate appropriately (typically 1 -12) in the Biosafety hood with
marker .
SAP lid labeling
SAP side labeling
o
oPrepare serial dilutions of serum . Refer to SHI -SOP- 10002 to process
samples.
oPrepare 10 serials of 2 -fold dilutions. The highest dilution is 10 folds. Prepare
duplicates. See step 5
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
10 20 40 80 160 320 640 1280 25600
D1:6μl+56μl2%FBS medium.
D2-D9:30μldiluted samples +30μlmedium.
oTransfer proper medium (according to cell type, phenol red free
DMEM+2%FBS+1%P/S) to the 50ml reservoir to facilitate the use of a
multichannel pipette (each plate needs 3.3ml, a liquot 4ml for each plate)
oSkip the first column. The outer wells are not used.
oColumns #3 -11 add 30 µl DMEM+2%FBS+1%P/S.
oNotes: Columns 3 -10, add 30 µl DMEM+2%FBS+1%P/S for 2 -fold serial
dilution from column 3. Column #11 on the 96W plate is control without any
antibody.
oColumns #3 -10, add 30 µl DMEM+2%FBS+1%P/S for 2 -fold serial dilution
from column 3
oColumn #1 and 12, add 60 µl DMEM+2%FBS+1%P/S.
FDA-CBER-2021-5683-0142299
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 10
oColumn #2 add 54 µl DMEM+2%FBS+1%P/S, then mix with 6 µl serum. Mix
without creating bubbles. Note: th e starting dilution is 1:10, then followed
with 10, 20, 40, 80, 160, 320, 640, 1280, 2560 dilutions (refer to step 4).
Overall dilution will be 1:20 once virus is added.
oColumn #2, mix using an electronic 12 -channel pipette (settings, P/M, 30/50,
speed 6/ 4, 5X), discard the tips
oLoad new tips, transfer 30 µl from column #2 to column #3 (settings, P/M,
30/50, speed 6/4, 5X), discard the tips
oRepeat step 12 for column #4 to #10
oAttention: remember to take 30 µl from column #10 after dilution and discard.
The total volume of the dilute serum should be 30µl/well
oSera dilution plate set -up(SAP) . Samples are run in duplicates. The outer
wells are not used for dilutions, just add media. Column 11 is reserved for
viral input (wells B11 -G11). Column 12 is reserved for cell control to monitor
cell contamination (wells B12 -G12) . Row H is reserved for a QCS (wells H2
H10). The QCS is used to assess plate and run suitability.
Well 1 2 3 4 5 6 7 8 9 10 11 12
Reciprocal
Serum
DilutionN/A 20 40 80 160 320 640 1,280 2,560 5,120 N/A N/A
A
Media + VirusMedia + Virus
B Test Sample 1 (Virus+Serum)
Virus input control
Cell controlC Test Sample 1 replicate (Virus+Serum)
D Test Sample 2 (Virus+Serum)
E Test Sample 2 replicate (Virus+Serum)
F Test Sample 3 (Virus+Serum)
G Test Sample 3 replicate (Virus+Serum)
H Quality Control Sample (Virus+Serum) Media + Virus
9.2.2. Processing of SA Ps
oTransfer SAPs and ROPs to BSL3.
oNote: Ref er to Tables 3 -9 of SHI -TM-10011-FM01.
oEnsure the incubators are reading at 37°C/5% CO 2. Confirm that water
reservoir is full.
oConfirm a correct set of assay plates.
oInspect plates to confirm that all wells of each SAP contain approximately 30
µL liquid and that volumes are consistent from well to well. Use the Atypical
Event(s) box (Table 13 of SHI-TM-10011 -FM01) to record the location of any
wells that have no liqui d or appear to have a volume other than 30 µL. Do not
use a pipette to measure well volumes; this step is a visual inspection only.
FDA-CBER-2021-5683-0142300
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 11
oNumber each SAP with a marker. The hand -written number dictates the
order in which the SAPs will receive a reagent and cor responds to its
respective ROP.
oPlace ROPs in incubator.
9.2.3. Preparation of Viral Stock Solution
oNote: Refer to Table 7of SHI- TM-10011 -FM01.
oPerform all manipulations of reagents and active virus in a Level II biosafety
cabinet within the BSL -3 laboratory using sterile techniques.
oIn BSC add 30 μl diluted SARS -CoV2 -mNG virus. 1µl reporter virus + 29µl
media/well (phenol red free). Any new batch of virus should be tested before
use. Use the volume of virus that can cause about 30% infection rate at 24 h
post- infection.
oComplete Viral Stock Solution (VSS) Table; verify with another analyst.
oRemove the appropriate number of frozen viral stock vials from -80 °C
freezer.
oThaw virus in distilled water at room temperature.
oIf multiple vials are needed, pool all vi als into a larger tube and pipette up and
down 3 times to ensure proper mixing.
oLabel a sterile bottle “VSS”. Add the appropriate volume of Assay Media.
oAdd the appropriate volume of SARS -CoV-2-mNG into VSS bottle and gently
swirl to mix.
9.2.4. Addition of Vira l Stock Solution to SA Ps and Neutralization of Virus
oTransfer diluted virus to the 25ml reservoir to facilitate the use of a
multichannel pipette (each plate needs ~2ml, 60 wellsx30µl=1800µ, always
make 1ml extra to the total volume to avoid running out)
oAdd 30μl of diluted virus to each well of the plate which containing diluted
sera (SAP) . Mix using an electronic 12 -channel pipette (settings, P/M, 30/50,
speed 6/4, 5X), after each column discard the tips
oIncubate at 37°C /5% CO 2for 1 hour.
oRecord the start and end times for this incubation in Table 8 in SHI -TM-
10011 -FM01.
9.2.5. Transfer Neutralization Reaction onto ROPs and Decontamination of
SAPs
oNote: Refer to Table 9of SHI- TM-10011 -FM01.
oRemove ROPs from incubator.
oTransfer 50μl virus -Ab diluted plate (SAP) to 96 -well plate seeded on day 0
(ROP) (plates containing cells 1.2x104 in 50 μl/well, after adding the diluted
reporter virus the total volume of each well will be 100µl). Mix using an
electronic 12 -channel pipette (settings, P, 50/80, speed 3/3, 3X), after each
column discard the tips. Note: do not touch the bottom of the plate, add the
mixture on the side of the well, make sure the pipette tip is touching the media
to avoid making bubbles when mixing. It is very important to avoid bubbles,
mix well, and do not disturb the cells to get an even infection.
FDA-CBER-2021-5683-0142301
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 12
oROP. Cells+Ab+reporter plate set up (black plate)
12 3 4 5 6 7 8 9 10 11 12
A
Sample 1 B +V +V +V +V +V +V +V +V +V +V Novirus
Sample 1 C +V +V +V +V +V +V +V +V +V +V Novirus
Sample 2 D +V +V +V +V +V +V +V +V +V +V Novirus
Sample 2 E +V +V +V +V +V +V +V +V +V +V Novirus
Sample 3 F +V +V +V +V +V +V +V +V +V +V Novirus
Sample 3 G +V +V +V +V +V +V +V +V +V +V Novirus
H
oIncubate ROPs at 37°C 5% CO 2for 16 -24HR.
oRecord the time the ROPs were placed in incubator in Table 9 of SHI-TM-
10011 -FM01.
9.3. Day 2: A cquire images
oNote: Refer to Tables 10 -12 of SHI-TM-10011-FM01.
oAt 16 -24 h post -infection, read the plate using Cytation 7 Cell Imaging Multi -
Mode Reader
oPrior to imaging, carefully examine the instrument CytationTM 7, bioStackTM
4 robotor and the workplace. Please ensure no used plates or any boxes
blocking the plate carrier.
oPower on the instrument, wait about 5 minutes to warm up the instrument.
Green light will show on the machine if instrument if it is ready. Open the
Gen5 software from the instrument attached computer and make sure the
software communicates with the instrument. Choose the right program for
reading (Since all the reading will be performed with plate lid -on, please make
sure the lid has been selected in your program).
oIf the bioStackTM 4 is going to be used for your experiment, place test -plates
(set of empty plates dedicated for testing only) on the bioStackTM 4 robotor
FDA-CBER-2021-5683-0142302
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 13
and run a quick test to verify the system and protocol. Ensure the
bioStackTM 4 robotor transfers the plates smoothly.
oIf the instrument is under any abnormal conditions, do not use the instrument.
Notify the lab manager or PI immediately.
oWeekly System Checks and Calibration plate instructions are described in
SHI-SHOP -10006
9.3.1. Preparation and A ddition of Hoechst 33342 Solution to ROPs
oCarefully transfer the plates in a secondary container from the incubator to
the biosafety cabinet.
oAdd 25 μl diluted Hoechst 33342 Solution (500 -fold in HBSS) to each well of
the 96 -well plates.
oSeal the plates carefully with films.
oIncubate plate at 37°C for about 20 minutes.
9.3.2. Image A cquisition and Counting on the Cytation 7
oAfter surface decontamination with calvicide, carefully transfer the plate with a
secondary container to CytationTM 7 or BioStackTM 4 to acquire images by
following the operation manual attached to the instrument.
oThe individual who performed scanning and counting completes the “Cytation
7 Analyst” section of Table 1 of SHI-TM-10011-FM01andenters the date that
these steps were completed.
oAt the end of the experiment, remove the plate from the carrier and transfer it
to the biosafety cabinet. Use 70% ethan ol to wipe out the plate carrier. And
power off the instrument. Disinfect and dispose all the plates and liquid
properly according to the laboratory waste disposal procedures.
9.4. Analysis of Data and Calculation of SA RS-CoV-2_NT Titers
oThe individual who perf orms data analysis completes the “SAS Operator/Data
Analyst” section on Table 1 of SHI-TM-10011 -FM01 and enters the date the
steps were completed.
oSARS -CoV-2_NT serum titers are calculated, and data is interpreted
according to the instructions outlined in SHI-SOP- 10011
oComplete Table 14 of SHI-TM-10011 -FM01.
9.5. SARS-CoV-2_NT Data and Document Review
oThe individual who performs data and document review completes the
“Reviewer” section on Table 1 of SHI-TM-10011-FM01 and enters the date
the steps were completed.
oAssembly of SARS -CoV-2_NT data packages, review of worksheets to
confirm that proper documentation practices are followed, and review and
processing of data are described in SHI -SOP -10012. Checklists and
comments for the documentation reviews are provided in SHI-TM-10011 -
FM01 Tables 15 and 16.
FDA-CBER-2021-5683-0142303
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies to
SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 14
10.REFERENCES
Xie X, Muruato M, Lokugamage KG et al, An Infectious cDNA Clone of SARS -CoV -2. Cell
Host & Microbe 2020, 27 (5): 841 -848.
Table 6. Form References
Document Title
SHI-SOP- 10011 NT50 and NT90 calculations for SARS -CoV-2
Neutralization Assay
SHI-SOP -10012 Data transferring between UTMB and Pfizer
SHI-SOP -10006 Equipment Management
SHI-TM-10011 -FM01 Manual 96 -well SARS -CoV-2 Neutralization Assay
Worksheet
SHI-TM-10011 -FM02 Manua l Sample Preparation for the 96-well SARS -
CoV-2 Neutralization Assay W orksheet
11.DOCUMENT VERSION MODIFICA TIONS
Table 7. Detailed Changes
List detailed changes for documents List rationale for each change
N/A N/A
FDA-CBER-2021-5683-0142304
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies
to SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 15
Document A pproval Record
SHI-SOP -10011
Manual 96 -well Neutralization Assay for the Detection of
Functional Antibodies to SARS -CoV-2 in Test Serum using
Cytation 7 Image Reader
Signed By: Date Signing Capacity
Fontes, Camila 20200 614 Author Approval
Shi, Pei -Yong Final ApprovalDocument Name:
Document Title:
FDA-CBER-2021-5683-0142305
UTMB -Shi Lab SOP
TITLE: Manual 96 -well Neutralization Assay for the Detection of Functional Antibodies
to SARS -CoV-2 in Test Serum using Cytation 7 Image Reader
Doc. Number: SHI -SOP -10011 Version: 2.0
UTMB -Shi Lab CONFIDENTIAL
Page 16
PERSONS TRAINED IN THIS PROTOCOL:
The following persons state that they have read and understood the protocol and the risk
assessment and have been properly trained in the procedures.
Name: Date: Signature: Name and Signature of
Trainer:
FDA-CBER-2021-5683-0142306