Slide 1

Biomarker Analysis in Serum, Plasma and Blood spots

Laure Moller, Ph.D.

Director Scientific Support

Meso Scale Discovery

Slide 2

Considerations for Biomarker Assays

• Pre-analytical factors

• Sample collection

• Method

• Time

• Sample processing

• Sample storage and shipment

• Assay performance

• Sensitivity

• Reproducibility

• Dynamic range

• Specificity

• Long term studies

• Lot to lot consistency

Slide 3

Dried Blood Spots

Advantages

• Small blood volume

• Minimal Processing

• Reduced shipping cost

• Field or in-home collection

• Less invasive

• Less storage space

Considerations

• Collection paper and method

• Analyte stability

• Storage conditions

• Release of analytes from whole bloodcomponents (different thanserum/plasma)

• Drying can damage some analytes

• Hematocrit can affect blood dispersalon the blotting paper - viscosity ofblood can differ since the amount ofred blood cells to plasma

• Small quantity of sample may limitdetection of low abundance analytes

• Capillary vs. venous bloodhttp://prohealthinsight.com/diseases-and-conditions/hematologic-diseases/what-is-anemia-and-its-causes/

Slide 4

Establishing Stability of Biomarker

• Collect whole blood via venipuncture

• Pipette 50 µL whole blood to the filter paper. Allow to dry

• Place one set of DBS in the freezer (-30°C or lower). “baseline” concentrationsof the analyte

• Expose other samples to

• 4 °C, room temperature, 37°C and one oscillating condition (12 h at 32°Cand 12 h at 22°C the presence of desiccant in a gas impermeable bag,for varying lengths of time up to 4 weeks

• Store in the same freezer as the baseline samples exposure time iscompleted.

• Analyze baseline and exposed samples together, on the same assay plate ifpossible, to maximize comparability.

Thomas W. Mcdade Development and Validation of Assay Protocols for use with Dried Blood Spot SamplesAmerican Journal Of Human Biology 26:1–9 (2014)

Slide 5

Sample Processing for DBS

Extraction

• Number of punches

• Size of punches

• Buffer composition

• Protease inhibitors?

Analysis

• Protocol (in-well extraction or off-line extraction)

• Incubation time

• Shaking speed

Slide 6

Number of Blood Spot Punches Required

• What is the biomarker concentration range in blood?

• What is the sensitivity of the assay?

• What is the extraction volume?

• What is the volume of sample required in the assay i.e. final dilution?

One 3.2-mm disc punched from a dried blood spot will contain approximately theequivalent of 1.4 μL of serum

For example – if the assay requires 100 µL of sample, extracting 1 x 3.2 mmpunch from a blood spot will result in a ~ 70 fold dilution of the sample.

Is the analyte measurable at that dilution or do you need to include more bloodspots or larger discs in the extraction?

Slide 7

Extraction Buffers

Some examples of extraction buffers for protein biomarker extraction from DBS

• PBS + 0.1% Triton + 0.03% Tween-20 + protease inhibitors

• PBS-T

• PBS containing 0.05% Tween and 0.5% BSA

• Assay diluent provided with kit

Slide 8

Extraction Protocols

• Add DBS punches to

• 96 well polypropylene plate or

• deep well plate,

• or filter plate

add extraction buffer, seal and shake

• Add DBS punches directly to MSD plates, add assay diluent

• Extraction times range from 1-2 h at RT or 4°C to overnight at 4°C

Slide 9

Calibrators

• Calibrators (Standards) should ideally be in a matrix that mimics the sample

• MSD provides assay diluents for this purpose for serum/plasma/CSF/urine inthe kits

• What has been done for DBS?

• Prepare calibrator in assay diluent and then mix with equal volume ofwashed erythrocytes to approximate a 50% hematocrit – mix gently byinversion

• Apply 50 µL drops to filter paper cards

• Allow to dry completely (e.g. overnight)

• Freeze in gas impermeable plastic bags with desiccant

Slide 10

Examples

Repeated measures of inflammation, blood pressure, and heart rate variability associated withtraffic exposures in healthy adults

Mirowsky, J. et al., Environmental Health 2015 14:66 DOI: 10.1186/s12940-015-0049-0

https://ehjournal.biomedcentral.com/articles/10.1186/s12940-015-0049-0

Capillary whole blood collected on a neonatal Guthrie card (903® Protein Saver Card, Whatman,Westborough, MA). The DBS were allowed to dry at ambient temperature away from sunlight, and thenplaced in a gas-impermeable plastic bag and stored at −80 °C. Blood samples were punched fromeach DBS and placed into individual wells of a 96 deep-well plate (USA Scientific, Ocala, FL). 200 μLof phosphate buffered saline (Sigma-Aldrich, St. Louis, MO) with 0.5 % Tween 20 (Sigma-Aldrich) wereadded to each well, completely submerging each sample. The plate was covered, cooled, andcontinuously shaken overnight (Model DS1, IKA Works). The following day, sample eluents wereanalyzed for protein content (C-reactive protein (CRP), serum amyloid A (SAA), soluble intercellularadhesion molecule (sICAM), soluble vascular adhesion molecule (sVCAM), interleukin 1-beta (IL-1β),interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α)) using Meso ScaleDiscovery.

Slide 11

Examples

Elevated Endogenous Erythropoietin Concentrations Are Associated with Increased Risk ofBrain Damage in Extremely Preterm Neonates

Korzeniewski, S.J. et al., 2015 PLoS ONE 10 (3): e0115083. doi:10.1371/journal.pone.0115083.

http://dx.doi.org/10.1371/journal.pone.0115083

Drops of blood were collected on filter paper (Schleicher & Schuell 903). Dried blood spots were storedat -70°C in sealed bags with desiccant until processed.

Because the volume of blood spots can vary in ways that we cannot easily measure, each proteinmeasurement was normalized to mg of total protein.

IL-1beta IL-6, IL-6R, TNF-alpha, TNF-R1, TNF-R2 IL-8, MCP-1, MCP-4, MIP-1B, RANTES (CCL5), I-TAC, I-CAM-1,ICAM-3, VCAM-1, E-selectin, MMP-1, MMP-9, CRP, SAA, MPO, VEGF, VEGF-R1,VEGF-R2, and IGFBP-1.

Slide 12

Examples

Relationship between neonatal blood protein profiles and placenta histologic characteristics inELGANs.

Hecht JL, et al. (2011) Pediatr Research 69: 68–73.

https://www.nature.com/pr/journal/v69/n1/full/pr9201114a.html

Drops of blood were collected on Schleicher & Schuell 903 paper (Whatman International Ltd, FlorhamPark, NJ) allowed to air dry, and stored at −70°C in sealed bags with desiccators until processed.

For protein elution, the frozen dried blood spots (DBS) were punched using 12-mm disposable biopsyAcuPunch (Acuderm, Inc., Fort Lauderdale, FL). The punched paper specimen was submerged in300-μL PBS-based buffer containing 0.1% Triton ×100 (Sigma Chemical Co.-Aldrich, St. Louis, MO)and 0.03% Tween-20 (Fisher, Hampton, NH) vortexes for 30 s and incubated on a shaker for 1 h at4°C. The punched paper along with the buffer was then transferred over the filter of a SpinXtube (Corning Fisher), centrifuged at 2000 × g followed by collection of the filtered eluted blood.An additional wash of the paper punch was performed in 100 μL for a final elution volume of 400μL. The eluted blood samples were aliquoted and stored frozen at −70°C in barcoded air-tightmicrotubes (USA Scientific, Orlando, FL).

IL-1beta IL-6, IL-6R, TNF-alpha, TNF-R1, TNF-R2 IL-8, MCP-1, MCP-4, MIP-1B, RANTES (CCL5), I-TAC, I-CAM-1,ICAM-3, VCAM-1, E-selectin, MMP-1, MMP-9, CRP, SAA, MPO, VEGF, VEGF-R1,VEGF-R2, and IGFBP-1.

Slide 13

Additional References Featuring MSD Assays for DBS

• Leviton A, Kuban KC, Allred EN, Fichorova RN, O'Shea TM, Paneth N. Early postnatal blood concentrations of inflammation-related proteins and microcephaly two years later in infants born before the 28th post-menstrual week. Early Hum Dev. 2011 Feb17. View in: PubMed

• Leviton A, Fichorova R, Yamamoto Y, Allred EN, Dammann O, Hecht J, Kuban K, McElrath T, O'Shea TM, Paneth N.Inflammation-related proteins in the blood of extremely low gestational age newborns. The contribution of inflammation to theappearance of developmental regulation. Cytokine. 2011 Jan; 53(1):66-73. View in: PubMed

• Fichorova RN, Onderdonk AB, Yamamoto H, Delaney ML, Dubois AM, Allred E, Leviton A. Maternal microbe-specific modulationof inflammatory response in extremely low-gestational-age newborns. MBio. 2011; 2(1). View in: PubMed

• McElrath TF, Fichorova RN, Allred EN, Hecht JL, Ismail MA, Yuan H, Leviton A. Blood protein profiles of infants born before 28thweeks differ by pregnancy complication. Am J Obstet Gynecol. 2011 Feb 22.View in: PubMed

• Leviton A, Kuban K, O'Shea TM, Paneth N, Fichorova R, Allred EN, Dammann O. The Relationship between Early Concentrationsof 25 Blood Proteins and Cerebral White Matter Injury in Preterm Newborns: The ELGAN Study. J Pediatr. 2011 Jan 14. View in:PubMed

• and Placenta Histologic Characteristics in Extremely Low GA Newborns. Pediatr Res. 2011 Jan; 69(1):68-73. View in: PubMed

• Bose C, Laughon M, Allred EN, Van Marter LJ, O'shea TM, Ehrenkranz RA, Fichorova R, Leviton A. Blood protein concentrationsin the first two postnatal weeks that predict bronchopulmonary dysplasia among infants born before the 28th week of gestation.Pediatr Res. 2010 Dec 9. View in: PubMed

• Leviton A, Fichorova R, Yamamoto Y, Allred EN, Dammann O, Hecht J, Kuban K, McElrath T, O'Shea TM, Paneth N.Inflammation-related proteins in the blood of extremely low gestational age newborns. The contribution of inflammation to theappearance of developmental regulation. Cytokine. 2011 Jan; 53(1):66-73. View in: PubMed

• Cramer DW, Vitonis AF, Pinheiro SP, McKolanis JR, Fichorova RN, Brown KE, Hatchette TF, Finn OJ. Mumps and ovarian cancer:modern interpretation of an historic association. Cancer Causes Control. 2010 Aug; 21(8):1193-201. View in: PubMed

• Gadjeva M, Paradis-Bleau C, Priebe GP, Fichorova R, Pier GB. Caveolin-1 modifies the immunity to Pseudomonas aeruginosa. JImmunol. 2010 Jan 1; 184(1):296-302. View in: PubMed

Slide 14

Analyte Abundance Differences in Different Matrices

Normal samples tested in V-PLEX assays – observed concentrations documented in product insert

Slide 15

S-PLEX

• S-PLEX takes MSD’s proven electrochemiluminescence technology to thenext level of SENSITIVITY. These new assays allow you to quantitatepreviously unmeasurable baseline samples with fg/mL sensitivity and a widedynamic range while preserving your precious samples.

• Currently offered as a testing service

LLOD

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Slide 16

Microsampling

• Dried Blood Spots on filter paper

• Serum separator cards

• Capillary tubes

• Microtainer device

• Volumetric Absorbtive Microsampling Technology (VAMS™)

https://www.neoteryx.com/alternative-dried-blood-spot-cards

Slide 17

MSD Catalog Assays

StandardMany analytes

Multiple species

Singleplex or multiplexpanels

Takes 6-8 weeks tocreate a customized

multiplex

Good intra-lotreproducibility

V-PLEXUltimate performance

Analytically validated

Multiple species

Singleplex or multiplexpanels

Easy and fast tocustomize

Very good intra andinter-lot reproducibility

Ideal for longitudinalstudies

U-PLEXUltimate multiplexing

flexibility

Singleplex or multiplex

Easy and fast tocustomize

Very good intra-lotreproducibility

Slide 18

The U-PLEX Menu – Now With 176 Assays and 29 Combos

Human

• 74 Assays• 14 popular assay Combos*

Non-Human Primate

• 64 Assays• 8 popular assay Combos

Mouse

• 38 Assays• 7 popular assay Combos

Single analyte detection with U-PLEX AntibodySets on MSD GOLD Small Spot Streptavidin

Open Spots

• Develop in-house assays• Combine with MSD assays for ultimate

flexibility

Assays Assay Development

Slide 19

U-PLEX Technology Fundamentals

• U-PLEX plates

• Up to 10 spots, each spot activated tocapture a specific U-PLEX linker(represented by color)

• U-PLEX linkers

• 10 linkers, each designed to bind to aspecific spot on the U-PLEX plate

• U-PLEX reagents

• Assay reagents (e.g., MSD GOLDSULFO-TAG™ NHS-Ester)

• Antibodies, Calibrators

• Diluents

• Read Buffer T (4X)

Slide 20

U-PLEX Linker Coupling

Biotin-binding domain

Matched linkerand spot

One spot of a plate

Slide 21

Combine U-PLEX Antibodies

• Select your analytes and choose theappropriate antibodies

• Combine the antibodies to make one solution

• Up to 10 U-PLEX-coupled antibodies canbe pooled

• Do not combine U-PLEX-coupledantibody solutions that share the samelinker

• Final solution is used to coat plates

• One step, not 10

• Antibodies bind to their spots on the plate fromsolution

Slide 22

Multiplexing with U-PLEXTM Products

• U-PLEX plates

• 10-spot plates activated to capture specificU-PLEX linkers (represented by color)

• Linker-Coupled Capture

• Biotinylated molecules coupled to U-PLEXlinkers

• Add 50 mL of solution and the coupledantibodies bind to the correct spot

U-PLEX PlateU-PLEX LinkerCoupled Antibodies

Slide 23

Build Personalized Multiplexes in 3 Simple Steps

• Couple:• Combine antibodies and linkers

• No purification of reagents

• No complex washing

• Combine coupled antibodies

• Coat:• Simple 50 mL addition to plates

• Incubate for 1 hour to overnight

• Plates are ready to use or store for 7 days

• Complete (Run Assay):• Add 25–50 mL of samples for 1 hour

• Add 25 mL of detection antibody for 1 hour

• Read

Slide 24

The U-PLEX Platform is a High Quality, Component Based System

Components are tested for:• Specificity• Reproducibility• Sensitivity• Cross-reactivity• Real-time stability

Components are assembled to order, enabling MSDto rapidly create and deliver custom multiplexes

Over 10,000 different multiplex combinations areavailable

U-PLEX components are designed and developedunder ISO 13485 and manufactured under ISO9001 standards

MSD has extensive experience developing andmanufacturing U-PLEX components and assays

MSD has manufactured:• > 33,000 plates• > 75,000 vials of antibodies• > 95,000 vials of Linkers• > 44,000 vials of Calibrators

Slide 25

U-PLEX Reproducibility

Slide 26

Typical U-PLEX Performance

Slide 27

Design Your Personalized Multiplex

Visit www.mesoscale.com/u-plex

Slide 28

The V-PLEX Platform – Analytically Validated Assays

48 Human Assays48 Human Assays

24 NHP Assays24 NHP Assays

13 Mouse Assays13 Mouse Assays

12 Rat Assays12 Rat Assays

V-PLEX assays cover critical researchareas including inflammation,oncology, neurodegeneration,angiogenesis, and now the Th17pathway research

Slide 29

V-PLEX: Developed and Manufactured Under Rigorous QualityStandards

• Long-term studies require biomarker assays that are both highly accurate andreproducible

• Rigorous processes maximize consistency in results and confidence in data

Development andValidationDevelopment andValidation

• Strict designcontrol process

• Characterizedcritical reagents

• Optimized assayparameters

ManufacturingManufacturing

• Over 20 years’experienceproducing highqualityimmunoassays

• State-of-the-artmanufacturingfacility

Quality ControlQuality Control

• Functionalevaluationincluding dynamicrange, limits ofquantification,precision, andnon-specificbinding

Slide 30

V-PLEX Assay Development Follows Strict Design Control Processes

Calibration Curve Optimization

• Calibrators are evaluated against internal reference and availableNIBSC/WHO standards

Reproducibility and Precision

• Typical % coefficient of variation (CV) for controls are <10% for both intra-runand inter-run

Limits of Quantification

• Upper and lower limits of quantification (ULOQ and LLOQ) are verified forevery manufactured lot

Specificity

• Non-specific binding is typically <1% for all assays within a multiplex

Evaluation of Matrix Effects

• Validated for recovery and linearity in multiple sample types

Stability

• 30-month shelf life from the manufacture date

Slide 31

Lot-to-Lot Reproducible Results

• During development samples are run across multiple lots.

• Controls are used to maintain lot-to-lot reproducibility once product is released

LLOQ

LLOQ

IFN-

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Slide 32

Multi-Lot and Multi-Year Quality Control Test ResultsDemonstrate Suitability of V-PLEX Kits for Long-Term Studies

• Quality control test results for 78 independently tested V-PLEX Proinflammatory Panel 1 (human)Kit lots

• Testing was performed between May 2013 and September 2016

• Each data point represents the results compiled for a minimum of 5 plates Average Intra-plateConc. %CV

High Mid Low

hIFN-γ 3.1 2.1 2.1

hIL-1β 3.2 2.5 3.1

hIL-2 3.0 2.6 3.0

hIL-4 3.3 3.2 3.2

hIL-6 2.5 2.4 2.4

hIL-8 2.4 2.4 2.5

hIL-10 1.9 1.7 2.3

hIL-12p70 4.7 4.5 5.0

hIL-13 2.2 2.3 2.4

hTNF-α 2.8 2.7 4.0

% Recovery measurements for High, Mid, andLow Control samples across 78 kits

%R

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High

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Slide 33

Performance: Spike Recovery and Dilution Linearity

• The study of biomarkers requires analysis of many diverse sample types

• Matrix studies such as spike recovery and dilution linearity are part of our rigorousassay validation processes

Spike recovery experiments performed on multiple matricesduring validation of the V-PLEX Human Vascular Injury Panel 2

Slide 34

Pre-Configured V-PLEX Kits

Each kit contains:

• 1 pre-coated MULTI-SPOT® Plate

• Multi-analyte calibrator (except Aβ PeptidePanel 1 which includes individualcalibrators)

• Individual detection antibodies

• Diluents

V-PLEX Proinflammatory Panel 1(human) Kit

IFN-g IL-1b IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNF-a

Lyophilizedmulti-analyte

calibrator

Slide 35

V-PLEX Custom Panel: Sub-plex of Preconfigured Kit

IFN-g IL-1b IL-2 IL-4 IL-5 IL-6 IL-10 IL-12p70 KC/GRO TNF-a

Customer gets: Validated panel Individual detection antibodies

for flexibility Fast delivery Controls available Certificate of Analysis

V-PLEX MouseCustom Panel

Shipped in 1-3days

Proinflammatory Panel 1 (mouse)

Slide 36

IFN-g IL-1b IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNF-a

Eotaxin Eotaxin-3 IL-8 (HA) IP-10 MCP-1 MCP-4 MDC MIP-1a MIP-1b TARC

GM-CSF IL-1a IL-5 IL-7 IL-12/23 p40 IL-15 IL-16 IL-17A TNF-b VEGF-A

Human Assays

V-PLEX Custom Panel: Across Multiple Preconfigured Kits

Customer gets: 2 Validated panels Individual detection antibodies

for flexibility Fast delivery Controls available 2 Certificate of Analysis

Pricing is still on a per assaybasis even though it uses 2 kits.

ProinflammatoryPanel 1 (human)

Chemokine Panel 1(human)

Slide 37

Ordering V-PLEX

• V-PLEX Assays are available in Standard and Plus versions

• Large Fit-for-Purpose Panels• Human 46-Plex Biomarker Panel• Right-sized Biomarker Panels for optimal analytical performance• Customized panels delivered off-the-shelf• Any custom assay combination can be ordered

• Fast Delivery• Shipped within 1-3 days

V-PLEX kits include:• Blended calibrator• Individual detection antibodies• Diluents• Comprehensive product insert• Certificate of analysis

V-PLEX Plus kits include:• All components of V-PLEX kits, plus:• Controls• Wash buffer• Plate seals

Slide 38

Visit Our Website To Find Out More About MSD’s V-PLEX Products

• Learn more about V-PLEXdevelopment, manufacturing, andquality control

• Access over 130 publicationsreferencing V-PLEX products

• Easily order a V-PLEX pre-configuredpanel or obtain a quote for your owncustomized multiplex

• https://www.mesoscale.com/en/products_and_services/assay_kits/v-plex

Slide 39

Applications for Multiple Sclerosis

• TH-17 Cytokine panel

(IL-17A, IL-21, IL-22, IL-23, IL-27, IL-31, MIP-3α)

• Neuro-inflammation Cytokine panel

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Slide 40

U-PLEX® Human α-Synuclein Assay

• MSD’s new validated assay for measuringhuman α-Synuclein

• Developed in collaboration with the MJFF

• Validated according to “Fit-for-Purpose”methods and CLSI guidance documents

• Measures human α-synuclein incerebrospinal fluid, plasma, saliva, andserum

• Market-leading analytical performance

• Multi-site testing ongoing

• Simple protocol

Median LLOD(pg/mL)

0.900

LLOQ(pg/mL)

8.00

ULOQ(pg/mL)

6,800

Slide 41

U-PLEX Human α-Synuclein Assay: Validation in Five Matrices

Exp. Date:09Nov2015Update:

Slide 42

Alzheimer’s Disease and Parkinson’s Disease Biomarkers

Slide 43

Choose the product best suited to your needs

Slide 44

MESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT,QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, U-PLEX, S-PLEX, V-PLEX, STREPTAVIDIN GOLD, MESO, Parsec,www.mesoscale.com, SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), U-PLEX(design), S-PLEX (design), V-PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC.

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