Document text
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 1
Title: Qualification Report for a Single -plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS- CoV -2 RBD Protein in Human Sera
Study Number: N/A
Parent Compound Number(s): PF-07302048
Alternative Compound Identifiers: N/A
Pfizer Vaccine Research and Development
401 N. Middletown Rd.
Pearl River, NY
090177e194a89e8a\Approved\Approved On: 18-Aug-2020 23:10 (GMT)
FDA-CBER-2021-5683-1072808
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 2Title: Qualification Report for a Single -plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS -CoV -2 RBD Protein in Human Sera
PRINCIPAL INVESTIGATOR: Guy Singh , Principal Sc ientist
CONTRIBUTING SCIENTIST (S):Darryl Charles, Estee Dilli , Nathaniel Garcia
PREPARED BY:
Guy Singh
Principal Scientist, Clinical and Diagnostic Assay Development
APPROVED BY:
Danka Pavliakova
Senior Principal Scientist, Clinical and Diagnostic Assay Development
Warren Kalina
Director, Clinical and Diagnostic Assay Development
Charles Tan
Senior Director, Earl y Clinical Development Biostatistics
David Cooper
Executive Director, High -throughput Clinical Immunoassay s & Diagnostics
Michael Pride
Executive Director, Clinical and Diagnostic Assay Development
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FDA-CBER-2021-5683-1072809
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 3Title: Qualification Report for a Single -plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS- CoV -2 RBD Protein in Human Sera
SYNOPSIS
This report documents the results of the statistical anal yses from assay qualification
experiments of a single -plex direct L uminex immunoassay for q uantitation of human IgG
antibodies to the receptor binding domain (RBD) of the spike (S) protein of severe acute
respiratory disease coronavirus 2 (SARS -CoV -2). The performance parameters examined in
the assay qualification were linearit y, precision and standard curve bias . From
these data, the assay range, lower limit of quantitation, and descriptive statistics of assay
parameters were determined. Key qualification outcomes are highlighted in the table below.
Key Qualification Outcomes
Antigen Assay Range Lower
Bound Plate Well
IgGConcentrat ion
(U/mL)aAssay Range Upper
Bound Plate Well
IgGConcentration
(U/mL)aLLOQbSample
Dilution Adjusted
IgGConcentration
(U/mL)aAssay Precision
c
COV RBDd0.002 301 0.128 000 1.1505
a.Units/mL .
b.Lower Limit of Quantitation.
c. .
d.Receptor binding domain of the spike protein of severe acute respiratory disease coronavirus 2.
The data provided in this report support the qualification of the single -plex dLIA for
quantitation of human IgG antibodies that bind to the RBD protein of SARS -CoV -2 and
confirm tha t the assay is suitable for its intended use when performed in accordance with
standard operating procedures by qualified personnel.
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FDA-CBER-2021-5683-1072810
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
LIST OF FIGURES ................................ ................................ ................................ ................... 6
1. OBJECTI VES ................................ ................................ ................................ ........................ 7
2. INTRODUCTION ................................ ................................ ................................ ................. 7
3. GLOSSARY ................................ ................................ ................................ .......................... 8
4. EXPERI MENTAL OUTL INE................................ ................................ ............................... 9
4.1. Materials and Methods ................................ ................................ .............................. 9
4.1.1. Methods ................................ ................................ ................................ ........ 9
4.1.2. Critical Reag ents................................ ................................ ........................... 9
4.2. Experimental Design ................................ ................................ ............................... 10
4.2.1. Linearity ................................ ................................ .................... 10
4.2.2. Precision ................................ ................................ ................................ .....11
4.2.3. Standard Curve Bias ................................ ................................ ................... 11
5. STATI STICAL METHO DS................................ ................................ ................................ 11
5.1. Sample Results ................................ ................................ ................................ ........ 11
5.2. Statistical Analy ses................................ ................................ ................................ .11
5.2.1. Linearity ................................ ................................ .................... 12
5.2.2. Precision ................................ ................................ ................................ .....12
5.2.3. Standard Curve Bias ................................ ................................ ................... 13
5.2.4. Assay Range ................................ ................................ ............................... 13
5.2.5. Assay Precision (Intermediate Precision) ................................ ................... 14
5.2.6. Sample Quantitation Range ................................ ................................ ........ 14
5.2.6.1. L ower Limit of Quantitation ................................ ..................... 14
5.2.6.2. Upper Limit of Quantitation ................................ ...................... 15
5.2.7. Assay Run Performance ................................ ................................ ............. 15
5.2.7.1. ................................ ................................ .15
5.2.7.2. Standard Curve Parameters ................................ ....................... 15
5.2.7.3. Quality Control Samples ................................ ........................... 15
6. RESUL TS AND DI SCU SSION ................................ ................................ .......................... 16
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FDA-CBER-2021-5683-1072811
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 56.1. L inearity Evaluation ................................ ................................ ............... 16
6.2. Precision Evaluation ................................ ................................ ................................ 17
6.3. Standard Curve Bias Evaluation ................................ ................................ ............. 18
6.4. Assay Range Based on L inearity , Precision, and Standard Curve
Bias ................................ ................................ ................................ ............................ 19
6.5. Assay Precision (Intermediate Precision) ................................ ............................... 19
6.6. Sample Quantitation Range ................................ ................................ ..................... 20
6.6.1. L ower Limit of Quantitation ................................ ................................ .......20
6.6.2. Upper Limit of Quantitation ................................ ................................ .......20
6.7. Assay Run Performance ................................ ................................ .......................... 20
6.7.1. ................................ ................................ .................. 20
6.7.2. Standard Curves ................................ ................................ .......................... 21
6.7.3. Quality Control Samples.............................................................................22
7. CONCLUSION ................................ ................................ ................................ .................... 22
8. DEVIATIONS ................................ ................................ ................................ ..................... 22
9. SUPPORTING DOCUME NTATION ................................ ................................ ................. 22
10. SUPPL EMENTAL INF ORMATION ................................ ................................ ............... 23
11. SUPPORTIVE FIGUR ES................................ ................................ ................................ ..24
12. SUPPORTIVE TABLE S ................................ ................................ ................................ ...25
LIST OF TABLES
Table 1. Terms and Definitions ................................ ................................ ................ 8
Table 2. SOPs and Robotic Methods within Scope of this Protocol ........................ 9
Table 3. List of Critical Reagents ................................ ................................ ........... 10
Table 4. Well Concentration Range Based on L inearit y Data .............. 16
Table 5. Well Concentration Range Based on Precision Data ............................... 17
Table 6. Well Concentration Range Based on Reference Standard Curve
Bias ................................ ................................ ................................ ........... 19
Table 7. Final IgG Assay Range – Well Concentration (Units/mL) ...................... 19
Table 8. ................................ 19
Table 9. Lower Limit of Quantitation (LLOQ) ................................ ...................... 20
Table 10. ................................ ................................ ................ 21
Table 11. Descriptive Statistics for Standard Curve Parameters.............................. 21
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FDA-CBER-2021-5683-1072812
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 6Table 12. Descriptive Statisti cs for QCS ................................ ................................ ..22
LIST OF FIGURES
Figure 1. Linearity Plot for RBD IgG dLIA ................................ ........... 16
Figure 2. Precision Plot for RBD IgG dLIA ................................ ............................ 17
Figure 3. Standard Curve Bias Plot for RBD IgG dLIA ................................ .......... 18
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FDA-CBER-2021-5683-1072813
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 7Title: Qualification Report for a Single -plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS- CoV -2 RBD Protein in Human Sera
Study Number: NA
Functional Area: Vacc ine Research and Development
Test Facility : Pfizer Vaccine Research, 401 North Middletown Road,
Pearl River, NY 10965
Study Initiation Date: 15Apr2020
Study Completion Date: 01Jul2020
1.OBJECTIVES
This report summarizes the methodology , results, and statistical anal ysis of the assay
qualification for a single -plex direct d LIA for q uantitation of IgG antibodies to the RBD
protein of SARS- CoV -2 in human serum , described in VR-TM-10294(see Section 9) Assay
qualification provides documented evidence that the assay , when performed in accordance
with standard operating procedures (SOPs) b y qualified personnel, is suitable for the
intended use to quantify RBD -specific IgG antibody concentrations in human sera.
2.INTRODUCTION
The single -plex SARS -CoV-2 RBD IgGdLIA measures specific IgG antibodies binding to
thereceptor binding domain (RBD) of the SARS -CoV-2spike protein (S) . This assay is
described in the test method, VR-TM-102 94 (see Section 9),and is based on the Luminex
MagPlex xMAP technology .
Magnetic, fluorescent Luminex microspheres are coated with according to
VR-SOP-LC-112951and then RBD protein. RBD coated
microspheres are added to serum samples, diluted in assay buffer and incubated, with
shaking, in a 96 -well microtiter assay plates for hours at °C. Unbound assay
components are removed by washing and a purified
anti-human IgG secondary antibody is added to the reaction wells. The secondary antibody
is incubated for minutes at °C. Unbound assay components are removed
by washing and the reaction is read on a Bio -Plex reader. The fluorescent protein coupled to
the secondary antibody allows measurement of the specific antibody bound to the antigen
coated microspheres. Fluorescen ce is expressed as median fluorescent intensities (MFI), and
the assay results are calculated against a reference standard with arbitrary assigned
concentration of 100.00 Units/mL . All sera are test ed in duplicate from independentl y
generated samples . Test samples are initially tested at dilutions,
dilutions are used to increase the likelihood that at least one result for an y
sample will fall within the useable range of the standard curve. Using acceptance crite ria
established in this report , failed plates and samples may be repeated.
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FDA-CBER-2021-5683-1072814
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 8The final assay results are expressed as the geometric mean concentration (GMC) of all
sample dilutions that produced a valid assay result within the assay range. GMC results tha t
are below the Lower Limit of Quantitation (LLOQ) are reported as Below Limit of
Quantitation (BLQ).
3.GLOSSARY
Table 1.Terms and Definitions
Term Definition
Assay Range Range of antibody concentrations that can be measured in the assay
plate w ell that have linearity, precision, and standard curve bias within the limits
described below . The assay range is generated from the most conservati ve lower and upper
well concentration limits based on linearity ,precision , and standard curve bias .
Beads Luminex Microspheres
BLQ Below Limit of Quantitation
CDAD Clinical and Diagnostic Assay Development
COV ID-19 Coronavirus Disease 2019
dLIA Direct Luminex ImmunoAssay
GMC Geom etric Mean Concentration
HCID High -throughput Clinical Immunoassays & Diagnostics
IgG Immunoglobulin G
LIMS Laboratory Information Management System
LLOQ Lower Limit of Quantitation -the lowest dilution -adjusted sample concentration that can be
determined with precision and falls within the linear portion of the assay
range .
MFI Median Fluorescent Intensity
mL Milliliter
NHP Non-human prim ate
QA Quality Assurance
QCS Quality Control Sample
RBD Receptor binding domain
RT Room Tem perature 18-25°C
S Spike glycoprotein of severe acute respiratory disease coronavirus 2
SARS -CoV-2 Severe acute respiratory disease coronavirus 2
Sample
Concentration antibody concentration calculated as the from the sample
that fall within the assay range and the lower and upper parameter limits of the
standard curve that is run on the same assay plate.
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FDA-CBER-2021-5683-1072815
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 9Table 1.Terms and Definitions
Term Definition
Sample Range Range of sample concentrations that can be quantified without
. The sample range is boun ded by the LLOQ and ULOQ. Results below LLOQ are
reported as BLQ. .
SOP Standard Operating Procedure
TM Test Method
ULOQ Upper Limit of Quantitation -the highest dilution -adjusted s ample concentration that can be
determined with precision and falls within the linear portion of the assay
range
VRD Vaccine Research and Development
Well
concentration antibody concentrations in the assay plate w ells
4.EXPERIMENTAL OUTLINE
4.1.Materials and Methods
Unique reagents prepared for this assessment are listed below, and all other materials are
described in the SOPs referenced in Table 2. Specific details regarding reagent catalog and
lot numbers and expiration, as well as the instrument identification numbers and maintenance
details w eredocumented in the assay worksheets wi thin data packages.
4.1.1. Methods
Table 2.SOPs and Robotic Methods within Scope of this Protocol
SOP Number Work Described in SOP Robotic Method Nam e
VR-TM-1029 4
(see Section 9)Test method N/A
VR-SOP-LC-112951Bead coating method N/A
VR-SOP-LC-111862Sample preparation using robot
VR-SOP-LC-106274Preparation of buffers N/A
4.1.2. Critical Reagents
The critical reagents, including antigen -coated microspheres (beads), reference standard,
QCS and PE -labeled secondary antibodies used in this assay qualification are listed in
Table 3.
The reference standard serum is a human sera (COVID-19
sera drawn at least day s after PCR -confirmed diagnosis from subjects
years of age) and assigned an arbitrary concentration of 100.00 U/mL of IgG antibodies
to the RBD antigen.
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FDA-CBER-2021-5683-1072816
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 10Table 3.List of Critical Reagents
RBD coated microspheres
Secondary antibody
Reference Standard Serum
COVID19 IgG STDa PCR confirmed Covid -19
sera
QCS1
COVID19 IgG QC1b
QCS2
COVID19 IgG QC 2c
QCS3
COVID19 IgG QC3d
a.Assay Standard.
b.Assay Quality control serum 1.
c.Assay Quality control serum 2.
d.Assay Quality control serum 3.
4.2.Experimental Design
The following sections describe the experimental designs for evaluating the
linearity , precision and standard curves.
4.2.1. Linearity
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FDA-CBER-2021-5683-1072817
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 114.2.2. Precision
Precision describes the closeness of measurements for a sample tested multiple times.
Precision is a measure of assay variabilit y that contains both repeatability and intermediate
precision. Repeatability measures the assay variability , usuall y within on e assay run,
whereas intermediate precision measures the variability within the same laboratory while
taking into account relevant sources of variation due to operating conditions ( eg,different
analysts, time, reagent lots).
The precision of the assay was examined using a panel of COVID -19
serawith IgG antibody concentrations to the RBD protein intended to span the expected
assay range and pre-pandemic human serum samples that have low or no specific IgG
to the RBD protein for a total o f samples . Refer to Supportive Table 12.2 for the
precision samples used.
All samples were tested in the assay at performed b y
a single
workstation, using the methods developed for clinical testing as
described in VR -SOP-LC-11186 .2 Refer to Supportive Figure 11.2 for the assay plate
layout used for the precision experiments.
Precision measurements ofthe samples were performed following VR-TM-10294
(see Section 9)over
. Results were anal yzed as described in Section 5.2.2 andSection 5.2.5 .
4.2.3. Standard Curve Bias
5. STATISTICAL METHODS
5.1.Sample Results
5.2.Statistica
l Analyses
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FDA-CBER-2021-5683-1072818
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 125.2.1. Linearity
5.2.2. Precision
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 135.2.3. Standard Curve Bias
5.2.4. Assay Range
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 145.2.5. Assay Precision (Intermediate Precision)
5.2.6. Sample Quantitation Range
The quantitation limits are defined as the sample concentration s that are between the LLOQ
and the ULOQ.
5.2.6.1. Lower Limit of Quantitation
The lower limit of quantitation (LLOQ) is the lowest sample concentration in the assay that
can be determined with precision and falls within the lin ear portion of the
assay range .
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 155.2.6.2. Upper Limit of Quantitation
The upper limit of quantitation (ULOQ) is the highest sample concentration that can be
determined with precision and falls into the linear assay range .
.
5.2.7. Assay Run Performance
5.2.7.1.
5.2.7.2. Standard Curve Parameters
5.2.7.3. Quality Control Samples
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 166. RESULTS AND DISCUSSI ON
Qualification data used for the results of the anal yses described herein are listed in
VR-MQR -10212-ATT01 (see Section 9).
6.1. Linearity Evaluation
Figure 1. Linearity Plot for RBD IgG dLIA
Table 4.Well Concentration Range Based on Linearity Data
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 176.2.Precision Evaluation
Figure 2.Precision Plot for RBD IgG dLIA
Table 5.Well Concentration Range Based on Precision Data
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FDA-CBER-2021-5683-1072824
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 186.3.Standard Curve Bias Evaluation
Figure 3. Standard Curve Bias Plot for RBD IgG dLIA
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 19Table 6.Well Concentration Range Based on Reference Standard Curve Bias
6.4.Assay Range Based on Linearity, Precision, and Standard Curve Bias
Table 7.Final Ig GAssay Range –Well Concentration (U nits/mL)
Antigen Linearity
RangePrecision
RangeStandard Curve
RangeFinal Assay Range
Lower Upper
COV RBDa0.002301 0.128
a.Receptor binding domain of the spike protein of severe acute respiratory disease coronavirus 2 .
6.5.Assay Precision (Intermediate Precision)
The intermediate precision of the assay was evaluated using the as described in
Section 5.2.5 and the results are summarized in Table 8.
Table 8. Variance Component Analysis of Sample Precision
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FDA-CBER-2021-5683-1072826
PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 206.6.Sample Quantitation Range
6.6.1. Lower Limit of Quantitation
The LLOQ is the lowest sample Ig Gconcentration in the assay that is precise
and falls into the linear assay range. Unlike the lower limit of the assay
range that is defined as a well concentration, the LLOQ is a dilution -adjusted sample
concentration. As described i nSection 5.2.6.1 , LLOQ is defined as the dilution -adjusted
lower l imit of the assay range. Table 9lists the LLOQ value for the RBD IgG dLIA .
Table 9.Lower Limit of Quantitation (LLOQ)
Antigen LLOQ (U/mL)
COV RBDa1.1505
a.Receptor binding domain of the spike protein of severe acute respiratory disease coronavirus 2 .
6.6.2. Upper Limit of Quantitation
6.7.Assay Run Performance
6.7.1.
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
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PFIZER CONFIDENTIAL
Page 21Table 10.
6.7.2. Standard Curve s
Descriptive statistics for the reference standard curves for RBD IgG dLIA are listed in
Table 11.
Table 11presents suitability limits on standard curves which were calculated as described in
Section 5.2.7.2 .
Table 11. Descriptive Statistics for Standard Curve Pa rameter s
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Page 226.7.3. Quality Control Samples
Descriptive statistics for the QCS samples from the qualification data for the RBD IgG dLIA
are listed in Table 12.
Table 12also shows QCS limits on the plates (refer to Section 5.2.7.3 ).
Table 12. Descriptive Statistics for QCS
7.CONCLUSION
The data provided in this repo rt support the qualification of the single -plex SARS -CoV -2
RBD IgGdLIA for quantitating RBD specific IgGin human sera .
The assay is suitable for
the intended use when performed in accordance with standard operating procedures b y
qualified personnel. The assay limits established from the data generated in this qualification
will be used to support assay suitability during clinical testing until such limits are refined in
a future validation.
8.DEVIATIONS
NA
9. SUPPORTING DOCUMENTA TION
1.VR-TM-10294, Single -plex Luminex Assay for Quantitation of IgG Antibodies to
SARS -CoV -2 RBD protein in Human Serum.
2.VR-MQR -10212 -ATT01, Supportive Data for VR- MQR -10212 .
3.VR-SOP-LC-11120, Data Review Procedures for Direct Luminex I mmunoassay s in
LIMS v6 .
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
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PFIZER CONFIDENTIAL
Page 2310. SUPPLEMENTAL INFORMA TION
1. VR-SOP-LC-11295, Preparation and Evaluation of Coated Microspheres
for use in Direct Luminex Assay s.
2. VR-SOP-LC-11186, Standard Operating Procedure for Running Method
Using
3.
4. VR-SOP-LC-10627, Preparation of Assay Buffers and Solution.
5. VR-SOP-FE-10111, BioPlex Sy stem Operatio n and Maintenance .
6. VR-DTN -11380, Planned Deviation from VR- SOP-FE-10111 Bio- Plex Operation
and Maintenance .
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
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PFIZER CONFIDENTIAL
Page 2411.SUPPORTIVE FIGURES
11.1. Plate Lay out for L inearity Experiments ................................ .......... 24
11.2. SARS -CoV -2 RBD IgG dLIA Assay Plate Lay out for Routine Testing ........................ 24
11.1. Plate Layout for Linearity Experiments
11.2. SARS -CoV -2 RBD IgG dLIA Assay Plate Layout for Routine Testing
The assay plate lay out in Supportive Figure 11.2 is used for routine testing.
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
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Page 2512.SUPPORTIVE TABLES
12.1. Linearity Samples ................................ ................................ .......................... 25
12.2. Sample Panel for Precision Evaluation ................................ ................................ ........... 26
12.3. Precision of COVRBD Well Titers Sample................................ 26
12.4. ................................ .................. 28
12.1. Linearity Samples
Sample Num ber Sample ID
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PFIZER CONFIDENTIAL
Page 2612.2. Sample Panel for Precision Evaluation
Sample Number ID Sample Number ID Sample Number ID Sample Number ID
12.3. Precision of COVRBD Well Titers Sample
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG Antibodies to SARS -CoV -2 RBD Protein in Human
Sera
VR-MQR -10212 , Ver. 2.0
PFIZER CONFIDENTIAL
Page 2712.3. Precision of COVRBD Well Titers Sample
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PF-07302048 : Qualification Report for a Single -plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV -2 RBD Protein in Human Sera
VR-MQR -10212 , Ver. 2.0
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Page 28
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Document Approval Record
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