Document text
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 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.Reag ents................................ ................................ ................................ .................... 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 RBD 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-2021-5683-0779569
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 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 RBD Coated Microspheres with Sera ................................ .12
9.5.Preparation of Plate Washer ................................ ................................ ......... 13
9.6. Secondary Antibody Preparation and Incubatio n ........................... 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 ................................ ................................ ................. 17
11.REFERENCES ................................ ................................ ................................ .................. 17
12.DOCUMENT VERSION MOD IFICAT IONS ................................ ................................ ..18
12.1. CRIF Number: VR -CRIF-20 -14620 ................................ ................................ .....18
LIST OF TABLES
Table 1. Chronological Summary of Previous Versions ................................ .......... 3
Table 2. Vaccine Research Functional Units ................................ ............................ 3
Table 3. Terms and Definitions ................................ ................................ ................ 3
Table 4. 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
Figure 1. Dilution Plate Diagram ................................ ................................ ............. 10
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FDA-CBER-2021-5683-0779570
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 31.HISTORICAL REVISION SUMMARY
Table 1.Chronological Summary of Pre vious Versions
Versio n
#Effective Date Originator Summary
1.0 Current Guy Singh New Document
2. PURPOSE
This p rocedure descri bes the method to quantitate Ig Gantibody levels to thereceptor binding
domain (RBD) of the spike (S) protein of severe acute re spirat ory disease coronavir us 2
(SAR S-CoV-2). The assay method measures IgGantibody levels for the RBD protein in
human serum.
3.SCOPE
Personnel in Vacci ne Research & Developme nt (VRD) that perform the s ingle-plex
SARS -CoV -2RBD IgGdLIAin regulated testing are required to follow t his test method .
Table 2.Vacci ne Research Functio nal Units
Functional Units Location
Vaccine Re search & Development Pearl River
4.GLOSSARY
Table 3.Terms and Definitions
Term Definit ion
Ag Antigen
Assay Plate The 96 -well flat -bottom opaque plate us ed to per form the as say.
Batch
Beads Aterm interchangeable with microspheres .
COV19 Coronavirus Di sease 2 019
Dilution Plate,
dLIA Direct Lumi nex Immunoassay
Expiratio n Date Expiration dates of r eagents will be assign ed based on the av ailable s tability data.
Specific expi ration dates may be recorded on reagen t labels, or within lab oratory
notebooks or documentation workboo ks refere nced by the reagent label.
IgG Immuno globulin G
Labware LIMS TheLabw are brand of a Lab oratory Information Management System whic h is a
database used to track specimens and their associated tests.
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FDA-CBER-2021-5683-0779571
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 4Table 3.Terms and Definitions
Term Definit ion
LNB Laboratory Notebook
Metadata Assay -relatedinformation th at is captured in LabW are LIMS. This i ncludes
information per taining to reagents, equipment and instrum ents used to complete the
assay.
MFI Median Fluorescent Intensity
QCS Quality Control Sample
QNS Quantity Not Sufficient. The design ation given w hen a s pecimen has insufficient
volume to complete testing.
RBD Recep tor bi nding domain
RT Room temperature defined as the ra nge from 18 to 25 °C
S Spike glycoprotein of s evere acute respirator y disease coronavirus 2
SAR S-CoV -2 Severe acute respirator y disease coronaviru s 2
VR-DWB Vaccine Research Document Workbook
WS Working Stock
5.GENERAL
Thesingle -plex SARS -CoV -2RBD IgGdLIAmeasur esSARS -CoV-2 RBD specific IgG
antibodies present in human serum sam ples.This assay isbased on the Luminex Mag Plex®
xMAP ®technolo gy pla tform. MagPlex ®microspheres are superparamagnetic carbox ylated
xMAP microsphere sand allow assay multiplexing and automation. The RBD protein is
and attached to coa ted microsphe res (VR-SOP-LC-11295).
RBD coated microspheres are added to the assay plates
with appropr iately diluted refe rence standard serum, QCSand unknown serum samples.
Theassay plates are incubated for hours at °C with shaking . After w ashing of
non-bound components, anti-human IgGsecondary antibody is added
to the microsphere mixture and incubated for mi nutesat RT . The fluorescent protein
coupled to the secondary antibody allows for measurement of the antibody bound to the
antigen coated microspheres by the Bio- Plex reader. Signals are expressed as median
fluoresc ent inten sities (MF Is) and read against a reference standard . The magnitude of the
fluorescent signal is proportional to the amount o f Ag-specific antibodies pres ent in the
sample.
6.RESPONSIBILITIES
All personnel and positions referred to in this proced ure are c onsidered to have an
alternate .
An alternate must ensure that they are trained and knowledgeable with the process.
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FDA-CBER-2021-5683-0779572
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
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Page 5Table 4.Roles and Responsibilities
Role Responsibilities
Analyst Performs the test method foll owing the p rocedures described in this SOP.
Com pletesdocumentation of the w ork perform ed and any deviations that occurred .
Follows applicable depar tmen talSOPs regarding safety , equipment use an d
documentation practices .
Initiates LDRs .
Notifies superviso r of any at ypical ev ents.
Supervisor or
designeeReviews and signs documentation.
Ensures that analysts have been trained on appropriate SOPs.
Ensures that equ ipment and instr uments have been p roperly calibrated and maintained.
Ensures LDRs are initiated and proper ly invest igated .
7.SAFETY
Biosafet y lev el 2 precautions should be taken when handling human sera. For current
guidelines refer to the CD C/NI H Biosafe ty in Microbiolo gical and Biomedic al
Laboratories Manual (BMBL), 5th Ed., 2009:
http://www.cdc.gov/biosafe ty/public ations/bmb l5/index.htm.
Personnel must be trained on current labora torysafet y procedures and made aware of the
Safety Data Shee ts (SDS) for chemicals being used, before perfo rming this 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 oratorysafet y guidelines con sult the Pearl Riv er 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/EQUIPMENT
All equipment and materials used in regulated testing must co nform to cu rrent dep artmental
guidelines for calib ration, maintenance and qualification.
8.1.Equipment
Bio-Plex Reader, Bio -Rad Cat. # orequivalent
Platform Shaker, or equivalent
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-2021-5683-0779573
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 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 pipe ttes, manual and electronic, vario us sizes
Pipetting Device, or equivalent
Vortex m ixer
Nutat ing m ixer
8.2.Materials
96-well flat bottom opaque white plates, or equivalent
Polypropy lene pipette tips, various sizes
Sterile disposable serological plastic p ipettes, various sizes
Dispos able polypropy lene tubes or container s, various sizes
Disposable light protective amber pol yprop ylene centrifug e tubes or conta iners, various
sizes
Aluminum sealing tape for 96 well plates
Reagent Reservoir s, various sizes
Polypropy lene micro snap cap tube s
Tube racks
Magnetic separators
Bioh azardous waste receptacle
Biohazard sharps waste containers
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FDA-CBER-2021-5683-0779574
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 78.3. R eage nts
8.3.1. Stand ardand QCS , Prepared as D escribed in VR-RGR- RS-10747
Standard reference serum: COVI D19 IgGSTD
QCS
QC1: COVI D19 IgGQC1
QC2: COVI D19 IgGQC2
QC3: COVI D19 IgGQC3
8.3.2. Microsp heres
Preparati on of antigencoated micro spheresis described in VR -SOP-LC-11295. Coated
microspher es are
8.3.3. Secondary Antibody
anti- human IgG (storeat
2to 8°C).
8.3.4.
8.3.5. Buffers
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FDA-CBER-2021-5683-0779575
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 89.PROCEDURE
secondary antibo dies are added to the plat es,incuba ted as described below and
plates are read on Bio -Plex instruments.
Any step of thi s procedure ma y be performed manually, or b y a qualif ied automated
method. Follow VR-SOP-LC-11186 for th e automated serum sample prepa ration by a
robo tic unit.
Incubat or temperatures stated in this method are temperature r anges as f ollows:
5°C = 2 -8°C; room temperature = 18 -25°C.
Bring all re agents and supp lies to ro om temperature before use in all of the following
steps. All assay steps a re to be performed at room tem perature unless otherwise
specified.
Movement of specim ens from a nd back to cold storage is logged using LIMS following
VR-SOP-LC-10774 . Forbatches i n LIMS the rea gent, equipment and incubation
informat ion will be captured as metadat a in LIMS . Raw data w ill be uploaded to LIMS.
VR-TM-10294-FM01 is the assay comple tion worksheet used when sample prep aration
is performed either manu ally or by automated m ethods. An as say completion worksheet
is not require dfor fully electronic /paperless batches where metadata is recorded in LIMS
by a robotic unit or manually .
VR-TM-102 94-FM02 is the worksheet used to prepare sample dilution plate s manually.
An assay completi on worksheet is not required for fully electronic/paperless b atches
where metad atais recor ded in LIMS.
Refer to VR -SOP-LC- 10601 for instruction on m aintaining the documentation of the data
package for paper -based docume ntatio n, if required. A Chain of Cus tody form is not
required for full y ele ctronic/paperless bat ches where a ll meta data and ra wdata is
recorded in/uploade d to LIMS .
Raw data CSV files will be t ransferred to the appropriate secure network drive upon
assay comple tion. Any other file t ypes do not need to be transferred, and raw da ta is not
required to be printed.
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FDA-CBER-2021-5683-0779576
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 99.1. P repa ration of Single-plex RBD Microspher es
The prepara tion of RBD antig en coated microspheres is described in VR-SOP-LC-11295.
9.2. P reparatio n of Sample Dilution Plate s
In the single -plex SARS -CoV -2 RBD IgGdLIA, each assay plate is derived from a
corresponding dilution plate. Each dilution plate contains three QCS, serial ly-dilute d
reference standard serum, blank and up to test samples. For ini tial testing, the
dilutions of test sera are . Additional dilutions can be evaluated if
needed. A dilution diagram of the plate is in Figure 1.
9.2.1. Robotic Preparation of Dilution Plates
Refer to VR-SOP- LC-11186 for sample and dilution plate preparation using the
robot.
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FDA-CBER-2021-5683-0779577
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 109.2.2. Manual Preparation of Dilution Plates
4.See Figure 1for the Dilution Pla te Dia gram.
Figure 1. Dilution Plate Diagram
9.2.2.1. Reference Standard
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FDA-CBER-2021-5683-0779578
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 119.2.2.2. Unknown S pecim enand QCS
9.2.2.3. Samples Requ iring Pre- dilutions
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FDA-CBER-2021-5683-0779579
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 129.2.2.4. Dilution Plate Completion
9.3.Preparation of Assa y Plates
9.3.1. Robotic Preparat ion of Assay Plates
Refer to VR-SOP-LC-11186for assay plate p reparation 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 RBD Coat ed Microspheres with Sera
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FDA-CBER-2021-5683-0779580
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 139.5. Preparation of PlateWasher
1.Prepare plate washer for use as described in VR -SOP-FE-10887. Record the
activit ies, 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. Seconda ry Anti body Preparat ion a nd Incubation
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FDA-CBER-2021-5683-0779581
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
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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-2021-5683-0779582
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
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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 empty
assay plates in a Bi ohazard waste receptacle .
9.10. Electronic Documentation
VR-TM-10294-FM01 is the assay completion worksheet used whensample pr eparatio n
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-10294-FM02 is the manual sample preparation workshe et used when a utomate d
sample preparation is not performed . An assay completio n worksheet is not required for
fully electronic /pape rless batches wh ere met adata is recorded in L IMS.
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FDA-CBER-2021-5683-0779583
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 16Refer to VR -SOP-LC-10601 for instruction on maintaining the documentatio n of the data
package, if required. A Chain of Custody form is not required for fully
electronic/paperless batches where all met adata andraw d ata is recorded in/uploaded to
LIMS .
For batches created in LIMS the reagents, equipment, and incubation informa tion will be
captured a s metadata in LIMS, therefore those s ections can be N/A if the worksheets are
used, and the raw data will be up loaded to LIMS.
Raw dat a CSV files will be tr ansferred to the appropriate secure network drive upon
assay completion. Any other file t ypes do not need to be transferred, and raw data is not
required to be printed.
9.11. Data Export and Primary Data Review
3.For LIMS runs, upload the data files back to LabWare LIMS a nd verify that all
equipment and reagent information for assay completion steps is accurately comple ted.
4. Complete the D ata Package chain of custody form, VR -SOP-LC-10601 -FM14 , if
required.
5. R eview a ssay documentation and/or LIMS metadata entry for accuracy andcompleteness
prior to submitting doc umentation f or secondar y review .
NOTE: If technical problem s occurred with the assay plate (plates are spilled or
dropped, etc.),
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FDA-CBER-2021-5683-0779584
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
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Page 1710.INTERPRETA TION OF RES ULTS
A qualified data reviewer anal yzes the data accordi ng to pre -specified acceptance criteria, as
descri bed i n VR -SOP-LC-1112 0.
11.REFE RENCES
Table 5.General References
Document Title
VR-SOP-QU-10004 Documen tation Pract ices in Vacci ne Research
VR-GWP -10041 Metadata Entry Pr ocedure in LabWare LIMS for Luminex Immunoassays
VR-SOP-QU-10726 Laboratory Devi ation Report
VR-SOP-LC-10788 Labeling and Traceability of Reagents and Solutions
Table 6.Form References
Form Title
VR-TM-10294-FM01 Assay Complet ion Worksheet for SARS -CoV -2RBD IgG dLIA
VR-TM-10294-FM02 Manual Sample Preparation Wo rksheet for SARS -CoV -2 RBD IgGdLIA
VR-SOP-LC-10601 -FM14 Gene ric L uminex Assay Data Package Chain ofCustody
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 Data Review Procedures for Dir ect Luminex I mmunoass ays Using Input Files or
LabWare v5 LIMS
VR-SOP-LC-1112 0 Data Review Procedures f or Direct Luminex Immunoassays in LIMS v6.
VR-SOP-LC-11186 Runni ng the Method Using Robot
VR-SOP-LC-11190 Standard Operating Procedure f or Ru nning Predilute Sample Method using
Robot
VR-SOP-LC-11295 Preparation of and Evaluation of Microsp heres for use in Direct
Luminex Assays
VR-RGR -RS-10747 Preparat ion 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-PlexSystem Operation and Maintenance
VR-SOP-LC-10586 Liquid Handling Techniques for Regulated Laborato ryProcedures
VR-SOP-LC-10601 Data P ackage Chain of Custody
VR-SOP-LC- 10774 Specimen Ma nagement Record Using LabWare LIMS
VR-SOP-FE-1088 7 Operation, Main tenance and Per formance Verificati on of
Microplate Washers
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FDA-CBER-2021-5683-0779585
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY
TITLE: Single -plex Luminex Assay for Qu antitation of
IgGAntibodi es toSARS -CoV-2RBD Protein in Human
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1812.DOCUMENT VERSION MOD IFICATION S
12.1. CRIF Number: VR-CRIF-20-14620
Table 8. Detailed Changes
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FDA-CBER-2021-5683-0779586
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FDA-CBER-2021-5683-0779587