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VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1
TABLE OF CONTENTS
LIST OF TABLES ................................ ................................ ................................ ..................... 2
LIST OF FIGURES ................................ ................................ ................................ ................... 2
1.HISTORICAL REVI SIONSUMMARY ................................ ................................ ..............3
2.PURPOSE ................................ ................................ ................................ .............................. 3
3.SCOPE................................ ................................ ................................ ................................ ...3
4.GLOSSARY ................................ ................................ ................................ .......................... 3
5.GENERAL ................................ ................................ ................................ ............................. 4
6.RESPONSIBILIT IES................................ ................................ ................................ ............4
7.SAFETY ................................ ................................ ................................ ................................ 5
8.MATERIAL S/EQUIPMENT ................................ ................................ ................................ 5
8.1. Equipment................................ ................................ ................................ ................. 5
8.2.Materials ................................ ................................ ................................ .................... 6
8.3.Reagents................................ ................................ ................................ .................... 7
8.3.1.Standard and QCS, Prepared as Described in VR -RGR-RS-10747............. 7
8.3.2.Microspheres ................................ ................................ ................................ .7
8.3.3.Secondary  Antibody ................................ ................................ ..................... 7
8.3.4. ................................ ................................ ................................ ...............7
8.3.5.Buffers................................ ................................ ................................ ..........7
9.PROCEDURE ................................ ................................ ................................ ........................ 8
9.1.Preparation of Single-plex S1 Microspheres ................................ ............................. 9
9.2.Preparation of Sample Dilution Plates ................................ ................................ ......9
9.2.1.Robotic Preparation of Dilution Plates ................................ ......................... 9
9.2.2. Manual Preparation of Dilution Plates................................ ....................... 10
9.2.2.1.Reference Standard ................................ ................................ ...10
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FDA-CBER-2021-5683-1072938
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 29.2.2.2. Unknown Specimen and QCS................................ ................... 11
9.2.2.3.Samples Requiring Pre -dilutions................................ ...............11
9.2.2.4.Dilution Plate Completion ................................ ......................... 12
9.3.Preparation of Assay Plates ................................ ................................ ..................... 12
9.3.1.Robotic Preparation of Assay  Plates................................ ........................... 12
9.3.2.Manual Preparation of Assay  Plates................................ ........................... 12
9.4.Primary Incubation of S1 Coated M icrospheres with Sera ................................ .....12
9.5.Preparation of  Plate Washer ................................ ................................ .........13
9.6.PE-conjugated Secondary  Antibody  Preparation and Incubation ........................... 13
9.7.Assay Completion ................................ ................................ ................................ ...14
9.8.Reading Assay  Plates on the Bio -Plex  I nstrument ................................ ...........14
9.9.Rereading of Assay  Plates Due to Errors ................................ ................................ 15
9.10.Electronic Documentation ................................ ................................ ..................... 15
9.11.Data Export and Primary  Data Review ................................ ................................ .16
10.INTERPRETATION OF R ESULTS ................................ ................................ ................. 16
11.REFERENCES ................................ ................................ ................................ .................. 17
12.DOCUMENT V ERSION MODIFICATIONS ................................ ................................ ..18
12.1.CRIF Number: VR -CRIF-20-14611................................ ................................ .....18
LIST OF TABLES
Table1. Chronological Summary  of Previous Versions................................ ..........3
Table 2. Vaccine Research Functional Units ................................ ............................ 3
Table3. Termsand Definitions ................................ ................................ ................ 3
Table4. Roles and Responsibilities................................ ................................ ..........5
Table 5. General References ................................ ................................ ................... 17
Table 6. Form References ................................ ................................ ....................... 17
Table 7. Specific References ................................ ................................ .................. 17
Table 8. Detailed Changes ................................ ................................ ...................... 18
LIST OF FI GURES
Figure1. Dilution Plate Diagram ................................ ................................ .............10
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FDA-CBER-2021-5683-1072939
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 31.HISTORICAL REVISION SUMMARY
Table 1.Chronological Summary of Previous Ve rsions
Version 
#Effective Date Originator Summary
1.0 Current Guy Singh New Document
2. PURPOSE
This procedure descri bes the method to quantitate Ig Gantibody levels to subunit 1 ( S1) of 
the spike (S) protein of severe acute respirator y disease coro navirus 2 (SARS-CoV-2).  The 
assay method measures IgGantibody levels for the S1protein in human serum.
3.SCOPE
Personnel in Vacci ne Research & Developme nt (VRD) that perform the s ingle-plex 
SARS-CoV-2 S1 IgGdLIAin regulated testing are required to follow this test method.
Table 2.Vaccine Research Functional Units
Functional Units Location
Vaccine Re search & Development Pearl River
4.GLOSSARY
Table3.Terms and Definitions
Term Definition
Ag Antigen
Assay Plate The 96-well flat-bottom opaque plate us ed to perform the as say.
Batch
Beads A term interchangeable with microspheres .
COV19 Coronavirus Di sease 2019
Dilution Plate, 
dLIA Direct Luminex Immunoassay
Expiration Date Expiration dates of r eagents will be assign ed based on the av ailable stability data.   
Specific expi ration dates may be recorded on reagen t labels, or within lab oratory 
notebooks or documentation workboo ks referenced by the reagent label.
IgG Immunoglobulin G
Labware LIMS TheLabware brand of a Lab oratory Information Management System whic h is a 
database used to track specimens and their associated tests.
LNB Laboratory Notebook
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FDA-CBER-2021-5683-1072940
FDA-CBER-2021-5683-1072941
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 5Table4.Roles and Responsi bilities
Role Responsibilities
Analyst Performs the test method foll owing the procedures described in this SOP.
Completesdocumentation of the w ork perform ed and any deviations that occurred .
Follows applicable depar tmental SOPs regarding safety, equipme nt use and 
documentation practices .
Initiates LDRs .
Notifies superviso r of any at ypical events.
Supervisor or 
designeeReviews and signs documentation. 
Ensuresthat analysts have been trained on appropriate SOPs.
Ensuresthat equipment and instruments ha ve been prop erly calibrated and maintained.
Ensures LDRs are initiated and proper ly investigated.
7.SAFETY
Biosafety level 2 precautions should be taken when handling human sera. For current 
guidelines refer to the CD C/NIH Biosafe ty in Microbiological and Biomedical 
Laboratories Manual (BMBL), 5th Ed., 2009: 
http://www.cdc.gov/biosafe ty/publications/bmb l5/index.htm.
Personnel must be trained on current labora torysafety procedures and made aware of the 
Safety Data Shee ts (SDS) for chemicals being used, bef ore performi ngthis procedure. 
For SDS information, refer to link at http:// .
All laboratory work is to be carried ou t incompliance with Pfizer safet y policies.  For 
general lab oratory safety guidelines consult the Pearl River Environmental Health and 
Safety Laboratory  Manual located at: 
http:/ .
Lab coats, safet yglasses and disposable gloves must be worn at all times when handling 
human specimens .
8.MATERIALS/E QUIPMENT
Allequipment and materials used in regulated testing must co nform to cu rrent departmental 
guidelines for calib ration, maintenance and qualification.
8.1.Equipment
Bio-Plex Reader, Bio -Rad Cat. #  orequivalent
Platform Shaker, or equivale nt
Scientific freezers, -80°C ±15°C and -20°C -15/+5°C
Scientific ref rigerator,2 to 8°C
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FDA-CBER-2021-5683-1072942
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 6 (magnetic bead) plate washer or equiva lent
Alarm timer
Single channel pipettes, various sizes
Multi-channel pipettes, manual and electronic, various si zes
  Pipetting Device,  or equivalent
Vortex m ixer
Nutating mixer
8.2.Materials


96-well flat bottom opaque white plates,  or equivalent
Polypropylene pipette tips, various sizes 
Sterile disposable serological plastic p ipettes, various sizes
Disposable polypropylene tubes or container s, various sizes
Disposable light protective amber pol ypropylene centrifuge tubes or containers, va rious 
sizes
Aluminum sealing tape for 96 well plates
Reagent Reservoir s, various sizes
Polypropylenemicro snap cap tube s
Tube racks
Magnetic separators
Biohazardous waste receptacle
Biohazard sharps waste containers
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FDA-CBER-2021-5683-1072944
FDA-CBER-2021-5683-1072945
FDA-CBER-2021-5683-1072946
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 109.2.2.Manual Preparation of Dilution Plates
Figure1. Dilution Plate Diagram
9.2.2.1.Reference Standard 
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FDA-CBER-2021-5683-1072947
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 119.2.2.2.Unknown Specimenand QCS
9.2.2.3.Samples Requ iring Pre- dilutions
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FDA-CBER-2021-5683-1072948
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 129.2.2.4.Dilution Plate Completion
9.3.Preparation of Assay Plates
9.3.1.Robotic Preparat ion of Assay Plates
Referto VR-SOP-LC-11186for assay plate preparation using the  
Robot.  Continue to Section 9.4to begin a ssay completion steps.
9.3.2.Manual P reparation of Assay Plat es
9.4.Primary Incubation of S1Coated Microspheres with Sera
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FDA-CBER-2021-5683-1072949
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 139.5. Preparation of  PlateWasher
1.Prepare  plate washer for use as described in VR -SOP-FE-10887.  Record the 
activities, as required, in the appropriate equipment logbook .
2.Ensure the Magnetic Bead Plate Adapto r is inserted for mag netic bead plate wash.
9.6.PE-conjugated Se condary Antibody Preparatio n and Incubation
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FDA-CBER-2021-5683-1072950
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 149.7.Assay Completion
9.8.Reading Assay Plat es on the Bio- Plex  Instrument
1.Prepare the Bio-P lex reader for use as described in VR -SOP-FE-10111.  Rec ord the 
activities, as required in the appropri ate equipment logbook.
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FDA-CBER-2021-5683-1072951
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 159.9. Rereading of Assay Plate s Due to Errors
6.Prepare assay  plates for disposal by  emptying the liquid off the plates.  Discard empt
y
assay plates in a Biohazard waste receptacle .
9.10.Electronic Documentation
VR-TM-
10293-FM01 is the assay  completion worksheet used w hensample pr eparation 
is performed either manually  or by automated methods.  A n assay completion worksheet 
is not required for fully electronic/paperless batches where metadata is recorded in LIMS.
VR-TM-10293-FM02 is the manual sample preparation workshe et used when a utomated 
sample preparation is not performed .  An assay  completio n worksheet is not required for 
fully electronic /paperless batches where metadata is recorded in L IMS.
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FDA-CBER-2021-5683-1072952
FDA-CBER-2021-5683-1072953
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1711.REFERENCES
Table 5.General References
Document Title
VR-SOP-QU-10004 Document ation Pract ices in Vacci ne Research
VR-GWP-10041 Metadata Entry Procedure in LabWare LIMS for Luminex Immunoassays
VR-SOP-QU-10726 Laboratory Devi ation Rep ort
VR-SOP-LC-10788 Labeling and Traceability of Reagents and Solutions
Table 6.Form References
Form Title
VR-TM-10293-FM01 Assay Complet ion Worksheet for SARS-CoV-2S1 IgG dLIA
VR-TM-10293-FM02Manual Sample Preparation Work sheet for SARS-CoV-2 S1 IgGdLIA
VR-SOP-LC-10601-FM14Generic Luminex Assay Data Package Chain of Custody
Table 7. Speci fic References
Document Title
VR-SOP-LC-10222 Procedure for M aking Sample Predilutions with  
Robot
VR-SOP-LC-10627 Preparation of Assay Buffers and Solutions for  Assays
VR-SOP-LC-11114 DataReview Procedures for Dir ect Luminex I mmunoassays Using Input Files or 
LabWare v5 LIMS
VR-SOP-LC-11120 Data Review Procedures f or Direct Luminex Immunoassays in LIM S v6.
VR-SOP-LC-11186 Running the Method Using  Robot
VR-SOP-LC-11190 Standard Operating Procedure f or Running Predilute Sample Method using 
 Robot
VR-SOP-LC-11295 Preparationof and Evaluation of  Microspheres for use in Direct 
Luminex Assays
VR-RGR-RS-10747 Preparation of Reference Standard Seru m and Quality Control Samples for 
SARS-CoV-2 S1 and RBD Direc t Luminex Immunoass ays
VR-SOP-FE-10111 Bio-Plex System Operation and Maintenance
VR-SOP-LC-10586 Liquid Handling Techniques for Regulated Laboratory P rocedures
VR-SOP-LC-10601 Data Package Chain of Custody
VR-SOP-LC-10774 Specimen Ma nagement Record Using LabWare LIMS
VR-SOP-FE-10887 Operation, Maintenance and Per formance Verificati on of  
Microplate Washers
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FDA-CBER-2021-5683-1072954
VACCINE RESEA RCH TEST METHOD PEARL RIVER, NY 
TITLE:Single-plex Luminex Assay for Qu antitation of 
IgGAntibodies toSARS-CoV-2 S1 Proteinin Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1812.DOCUMENT VERSION MOD IFICATIONS
12.1.CRIF Number: VR-CRIF-20-14611
Table 8. Detailed Changes
List detailed changes for document( s) 
Includesection number(s) for eachList rationale for each chan ge
New NA
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FDA-CBER-2021-5683-1072955
Document Approval Record
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