Powerpoint Template

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Application of Electrochemiluminescence on the
Meso Scale Discovery Platform Cell Based Assays
Monica Errico Scientific Services merrico_at_mesoscal
e.com
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Cell based Assays For Predicting Toxicological
Events

• Overview
• Overview of MSD Technology
• Examples of Cell Based Assays for predicting
  toxic events
• Intracellular signal transduction
• Cytokines as secreted biomarkers

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Traditional Methods for Predicting Toxicological
Events

• Traditional toxicity - utilizes a set of
  standardized animal-based studies that are used
  to predict if exposure to an experimental
  compound will result in adverse events in humans.
  
• Methods are low throughput
• Often fail to be predictive of human adverse
  events
• Do not provide insight into mechanisms of
  toxicity
• MSD Biomarker and Cell Based can be used to
• determine the mechanisms of toxicity

Injection of rodent model
Multiple time points, dosages, age, sex

• Evaluate behavior
• LD50 determination
• Histology

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Challenges for Cell Based Assays for Predicting
Toxicological Events

• Challenge
• Need an assay that can be applied from bench to
  bedside
• Cell models may require evaluation of multiple
  cell lines
• Ex vivo
• organotypic culture
• slice models
• In vivo
• Tissue/biopsy samples
• Bodily fluids
• May need to test multiple species
• Limited sample

• Solution
• Need a platform that works well in both simple
  and complex matrices
• Need very sensitive assays with a broad dynamic
  range
• Need assays that span multiple species
• Need a platform that can multiplex

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Cell based assays on MSD

• MSD Assays
• Small sample volume (10-25 ul)
• Ultrasensitive with a broad dynamic range (3-5
  log).
• Compatibility with complex matrices
• Simple protocols
• Multiplexingreduced sample volume, labor and
  cost
• MSD Platform
• Open platform for novel applications or
  development of assays
• Ability to read partial plates
• Automation-compatible
• Wide range of fully developed assays available
  (gt400).
• On site scientific support

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MSD Platform

• Available Kits
• Over 400 Kits (single analyte and multiplex)
• Complete with Plates, Diluents, and Standards.
• Diseased Focus
• Cost Effective
• Reduced Workflow
• Multiple species

• Electrochemiluminescence
• Large Dynamic Range
• High Sensitivity
• Complex Matrices
• Reduced Sample Volume
• Reduced Interference

• Robust Instrumentation
• 96 and 384-well Plates
• Multiplex Enabled
• Non-Fluidics Based Instrument.
• CCD Imaging System
• Validated in Regulated Environment

• Flexible Assay System
• Bare and Coated Plates
• Labeling Reagents
• Secondary Reporters
• QuickPlex Assays for Multiplex Development

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What is Electrochemiluminescence?
Electro-
chemi-
luminescence
electrochemically driven
chemical energy
emitting light
N
N
N hn
REDOX

• Selective
• Robust
• Stable
• No known instance of auto
• electrochemiluminescence

Ruthenium (II) tris-bipyridine NHS
ester SULFO-TAG
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Plate-based Electrochemiluminescent Assays
MULTI-ARRAY Plate

• Electrodes are built into the bottom of the plate
  and are energized within the instrument.
• Proximity only labels near electrode surface
  are detected, enabling non-washed or reduced
  washed assays.
• The electrochemical reaction occurs within the
  plate and light is measured through a CCD camera
  or photodiodes.
• Carbon surface with hydrophobic dielectric also
  forms physical and surface tension barrier

Counter electrode
Working electrode
Dielectric
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MSD Multiplex Plates

• Manufactured using deposition of conductive and
  insulating layers similar to computer circuit
  board manufacturing.
• Multiple electrical circuits within the same
  well.
• Each circuit is spatially independent.
• Biospecific reagents are deposited on each
  circuit or spot.
• Multiplexed assays are possible through the
  specificity of the capture reagents and the
  ability to resolve images.

384 wells 4 spots/well
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Large Dynamic Range Assays with High Sensitivity
1e006
Human TNF-a

• MSD Ultrasensitive TNF-alpha
• Detection Limits 0.25 pg/ml
• LLOQ 1-2 pg/ml
• Upper End 2500 pg/ml
• Sample 25 ul

100000
MSD Assay
Disease Human
Signal
10000
Sensitive ELISA
Normal Human
1000
Traditional ELISA
100
0.1
1
10
100
1000
10000
Concentration (pg/ml)
0.1
100
Concentration (pg/ml)
High Sensitivity ELISA
MSD Ultrasensitive
Unlike ELISAs, most MSD assays allow for
measurement of normal and disease populations
with a single dilution.
Typical ELISA
Detection Limit (pg/ml)
15-30
0.2
0.25
LLOQ (pg/ml)
20-50
0.5-1
0.5-2
Upper Limit (pg/ml)
200-1000
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10000
Sample Volume (ul)
200
50
25
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Similar Performance Across Spot Densities
96-well Single Spot
96-well 10-Spot
384-well 4-Spot
LOD 0.5 pg/ml
LOD 0.2 pg/ml
LOD 0.8 pg/ml
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MSD Catalog Items

• Research Kits
• Over 400 assays and multiplexes currently
  available through existing MSD catalog
• Qualified Kits
• Complete Assay Qualification and Lot Verification
• Custom Multiplex Assays
• Custom combinations of multiplexed assays.
• Prototype Assays
• Coating service and assays with customer provided
  antibody pairs.

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ASSAY DEVELOPMENT TOOLS

• REAGENTS
• Buffers
• Blockers
• DETECTION
• Sulfo-Tag NHS Ester
• Streptavidin Sulfo-Tag
• Anti-species Sulfo-Tag Antibodies
• CUSTOM COATING SERVICES
• Your antibodies coated onto plates using MSD
  manufacturing capabilities

• PLATES
• Bare (uncoated)
• Streptavidin/Avidin
• Anti-species (mouse, rabbit)
• Glutathione
• Protein A
• TECHNICAL ASSISTANCE/ADVICE
• Team of applications scientists
• RD Department

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No harsh coupling chemistry required
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The scope of cell based assays performed on MSD
Binding to cell surface targets
Receptor protein phosphorylation
Secreted proteins e.g. Cytokines
Internalization of drug product
Phosphorylation of intracellular substrates
Cell proliferation Cell death/apoptosis Differenti
ation
Intracellular trafficking
Gene expression
Protein expression
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Intracellular Signaling for Predicting
Toxicological Events

• ASSAY and Drug Development Technologies
• Volume 5, Number 3, 2007

ASSAY and Drug Development Technologies Volume 5,
Number 3, 2007
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Types of Intracellular Signaling Assays Developed
on MSD

• Total Receptor expression
• Receptor Phosphorylation
• Intracellular signal transduction

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MSD Intracellular Signaling Assays

• Traditional Methods
• Typically measured using western blots
• Negatives
• Very slow methods
• Qualitative
• Cannot be performed in a regulated environment
• MSD Phosphoprotein Assays
• Extremely reproducible results
• Fast- total assay time is 3.5 hours
• Quantitative can be validated
• Multiplexing allows for in-well controls

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MSD Intracellular Signaling Assays Work Well in
Complex Matrices

• Tumor lysates
• Tissue lysates (e.g. adipose, liver, brain)
• Xenografts
• Matrigel plugs
• Bone marrow biopsies
• Whole blood

p/t AKT and p/t GSK 3ß in human tumor xenografts
AKT
GSK-3b
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Application of MSD Cell Signaling Assays
Decision Making for Pathway Analysis
Oxidative Stress DNA Damage Apoptosis
Markers of Cell Viability/Survival
How Many Marker to Evaluate?
Cellular Stress HSP27 HSP70
Apoptosis Cleaved Caspase 3 Cleaved PARP Phospho
p53 Ubiquitinated MDM2
DNA Damage Phospho p53 Phospho Rb Phospho Histone
H3
Oxidative Stress/ Inflammation NFK-b c-Jun P38
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Intracellular Signaling in Safety Assessment
Akt Signaling
Akt in Toxicology Reference Kattla, J.J., Carew,
R.M., Heljic, M., Godson, C., Brazil, D.P.
(2008)  Protein kinase B/Akt activity is involved
in renal TGF-beta1-driven epithelial-mesenchymal
transition in vitro and in vivo. Am J Physiol
Renal Physiol. 295(1) F215-25.
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Catalog Item Simultaneous Detection of Phospho
and Total Akt

• Logathimically growing Jurkat cells (positive)
  were treated with LY294002 (50 nM, 2.25 hours)
  (Negative). Whole cell lysates were added ot he
  MSD MULTI_-SPOT 96-Well 4 Spot plates coated with
  anti-phospho-Akt antibody and anti-total Akt
  antibody on two of the four spatially distinct
  electrodes per well. Phosphoorylated and toal
  Akt were detected with anti-toal Akt antibody
  labeled with MSD SULFO-TAG reagent.

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Catalog Item Akt Signaling

• Logarithmically growing Jurkat cells were treated
  with PMA (200 mM, 15 min) (positive) or LY294002
  (50 uM) and staurosporine (1 uM, 2,25 hr)
  (negative).

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Create Custom Multiplex Panels
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Convert Existing ELISA

• Antibody pairs are readily available through a
  variety of sources.
• Capture antibodies are immobilized and
  streptavidin-SULFO-TAG is used as the detector.
• With little or no optimization the same regents
  as used in ELISA have improved sensitivity and
  dynamic range

Strepavidin-SULFO-TAG
Biotin Polyclonal Detection
Monoclonal Capture
MSD
ELISA
Range of ELISA
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Advantages MSD of Intracellular Signaling Assays

• Quantitative
• Sample conservation most assays require 10 ug
  of protein or less
• Faster time to results
• Intracellular signaling assays can be validated
  completely automatable
• Compatible with complex matrices cells, tumor
  biopsies, tissue lysates, whole blood

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Secreted Biomarkers as Cell Based Assays

• Secreted proteins
• Cytokines
• Growth factors
• Metabolic markers

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Cytokine Storm in Phase 1 Trial of TGN1412
Ganesh Suntharalingam, F.R.C.A., Meghan R. Perry,
M.R.C.P.,Stephen Ward, F.R.C.A., Stephen J.
Brett, M.D., Andrew Castello-Cortes, F.R.C.A.,
Michael D. Brunner, F.R.C.A., and Nicki
Panoskaltsis, M.D., Ph.D. Cytokine Storm in a
Phase 1 Trial of the Anti-CD28 Monoclonal
Antibody TGN1412, New England Journal of
Medicine. 2006 10.1056
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Cytokine Assays Large Dynamic Range Assays with
High Sensitivity
Human TNF-a
0.1
1
10
100
1000
10000
Concentration (pg/ml)

• Advantages
• Ultrasensitive (sub-pg/ml range) with a broad
  dynamic range (3.5-5 logs)
• Conserves samples MSD assays use 10-25 ul of
  sample
• Simple protocol typical assay time is 3-4.5
  hours.

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Effect of Mitogen /- drug action in T-cells

• Tissue Culture Protocol
• Add 25 uL of sample or standard and incubate 1-2
  hr with shaking.
• Add 25 uL of 1 ug/m detection antibody solution
  and incubate 1-2 hr with shaking.
• Wash with PBS 0.05 Tween 20
• Add 150uL of 2X MSD Read Buffer T, Read

PHA / PMA stimulation
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Responses to Drugs and Mitogens
IGF-1
Freebern, W.J., Haggerty, C.M., Montano, I.,
McNutt, M.C., Collins, I., Graham, A.,
Chandramouli, G.V.R., Stewart, D.H., Biebuyck,
H.A., Taub, D.D., Gardner, K. (2005)
Pharmacologic profiling of transcriptional
targets deciphers promoter logic. The
Pharmacogenomics Journal. 5(5) 305-323.
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Compatibility with Complex Samples

• Data shown is from one spot of a 7-plex plate,
  precious sample is conserved and measured at much
  higher throughput.
• Ability to analyze complex matrices including
• Serum /Plasma
• Cell lysates /Tissue Homogenates
• BAL
• Nasal lavage
• Sputum
• Synovial fluid
• Breath Condensate
• CSF
• Urine
• Tumor extracts
• Blood spots
• Vaginal secretions
• Tears
• Validated by clinical labs.

Brigham and Womens
Harvard Medical
School
Hospital
Fichorova et. al,Trichomonas vaginalis
Lipophosphoglycan Triggers a Selective
Upregulation of Cytokines by Human Female
Reproductive Tract Epithelial Cells INFECTION
AND IMMUNITY, Oct. 2006, p. 57735779
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Multiplexing secreted markers with MSD
Multi-Spot Plates

• MULTIPLEXING Panels
• Cytokines
• Metabolic
• Vascular injury
• Cardiac Injury
• Kidney Injury
• Growth Factors
• Hypoxia
• Bone
• Alzheimers disease
• For more information visit our website at
  www.mesoscale.com

1
2
A
B
Counter Electrode
Dielectric
Labeled-Ab
TNF-a
Capture-Ab
Working Electrode
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Summary

• The use of cell based assays will help us
  determine the mechanisms of toxicity.
• Understand the molecular mechanisms of toxic
  events in rodents and human models.
• Cellular models for toxicology can be implemented
  earlier in the discovery and development process
  allow us make important decisions about chemical
  matter and series at an earlier stage.
• The MSD Platform is a rapid, robust platform that
  allows for multiple measurements on a single
  sample
• Reducing labor and cost
• Reducing the samples requirement
• Improving the time to result
• MSD cell based assays have a broad dynamic range
  (3-5 log) and good sensitivity allows for
  quantitative measurements
• MSD Sector Imagers are simple operation
• fast read time (read time 1 min per plate)
• No fluidics and no routine maintenance
• 21CFR part 11 compliant
• Open platform for other applications (biomarkers,
  cytokines, immunogenicity)
• Use kits available off the shelf or develop your
  own assays
• MSD has developed and qualified several biomarker
  panels to study damage in several organ systems
  including Kidney, Muscle, Vascular, and
  Endocrine

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What can MSD offer You?

• Platform for Bioassays
• Replacement for ELISAs and Westerns
• Minimal sample consumption
• Compatibility with difficult matrices
• Validation for clinical applications (GLP)
• Simple assay protocols
• Multiplexing
• Kits and panels for single- and multiplex
  biomarkers
• Cytokines
• Phosphoproteins
• Clinical markers
• Strong collaborative relationship
• Resources
• Assay development
• Technical expertise

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