Transcript Slide 1

B U I L D I N G V A L U E, E N H A N C I N G L I V E S
Beadlyte multiplex assays/Luminex
Ciaran Sewter PhD, Upstate Ltd
Presentation Overview
Luminex – the background
Principles of the Luminex Technology
Beadlyte Cytokine Detection Assays
History of Assay Technology
Technology platform basics
– Industry advances in multiplex and parallel analysis
Test tubes
Microwell plate
Microarray
xMAP® Technology:
Microsphere-based
liquid array
What is a multiplex assay?
The ability to simultaneously detect multiple biomarkers or
intracellular signaling modifications
Antibodies are most often used in a sandwich immunoassay
fashion and are immobilized on chips as “planar arrays” or
conjugated to micro beads as “suspension arrays.”
Planar Array
Bead Array
Bioperspectives
The Use of the Luminex xMap Suspension
Array Technology in the Drug Discovery Process
Enzymatic
Immunoassay
Receptor-Ligand
Molecular Biology
• Over 75 peer reviewed articles on Luminex technology
• Becoming one of the fastest growing technology areas
Many Beadlyte® Kits are Bead-Based
Sandwich Immunoassays
1. Bead with capture antibody
2. Capture antibody binds analyte
3. Fluorescence labelled reporter
antibody binds to capture
analyte
Laser
A
Laser
B
4. Bead ID and reporter quantity
determined by laser detector
The flow cell – where it all happens..
Red laser reads the bead,
i.e. the target
Green laser detects the
amount of the target
Principles of the Luminex Technology
The Luminex machine
Data Capture
Software reports results in real-time
Up to 9600 results read in < 1 hour
Beadlyte Immunoassay format
for the Luminex Machine.
After
In
Free
The
assays
phycoerythrin
immune-complex/
excess
another
with
streptavidin-PE
wash
highisstep
analyte
excited
The
A
primary
biotinylated,
Microsphere
primary
antibody
antibody
analyte
is
specific
a
binds
Streptavidin
numbers
binds
by
microsphere
thetoreporter
the
excess
non-specifically
PE
is then
laser
isbiotinylated
added
excited
and
to
5.6mM
for
to
specific
the
the
specific
analyte
polystyrene
reporter
analyte
is
antibody
bead
–
no
thethe
reporter
bound
emits
by
assay.
a******
biotinylated
fluorescence
antibodies
The
laser.
biotinylated
reporter
The
bind
which
bead
tois
with
conjugated
crossreactivity
added
two
toemmission
fluorescent
thetoby
assay
the
with
bead
other
after
dyes
reporter
the
antibody
is
specific
quantified
Strep-PE
antibody
leading
in
the
ato
binds
nonLuminex
is
a
signal
to
incorporated
surface
analytes
another
by
wash
occurs.
an
into
step.
amine
it
in
one of the
specific
amplification.
reader.
quantified
manner.
by
four
the
available
luminex
different
coupling
ratios.
reaction
sites.the bead identified.
and
A standard curve is generated from a single
mixed standard…
Cytokine Concentrations (pg/ml) from
1 l of normal human basal tears
(sample analyzed in triplicate)
Beadlyte – Cytokine / Chemokine Detection
Sample Types
Sample Volumes
Culture supernatant
• Routine protocol = 50ul
Serum
- s/n = 50ul
- serum = 25ul serum
Plasma
25ul serum diluent
Tissue lysates
Urine
Blood spots
Gingival Crevicular Fluid
Nasal Lavage Fluid
Tears
Cerebral Spinal Fluid
Broncho-Alveolar Fluid
• Have modified low volume protocols
- 25ul, 10ul, 5ul, 1ul
Cytokine Assays Available from Upstate
Preconfigured Human & Mouse Multi-Cytokine Kits
Flexible Human (26) and Mouse (21) Detection Systems
–
(1 or 5 plate format)
Serum/Plasma Diluents
Preconfigured assays
Human:
Detection 1: IL-2, IL-4, TNFa, IFNg, GM-CSF
Detection 2: IL-1b, IL-6, IL-8, IL-10, IL-12 (p70), TNFa
Detection 3: IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 (p70), GM-CSF, INFg, TNFa
Detection 4: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IFNg, MCP-1, RANTES,
TNFa
22-plex: IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-12
(p40), IL-13, IL-15, IP-10, Eotaxin, IFNg, GM-CSF, MCP-1, MIP-1a,
RANTES, TNFa
Mouse:
Detection 1: IL-2, IL-4, IL-10, TNFa, IFNg
Detection 2: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), GM-CSF, IFNg, TNFa
Preconfigured kit components
Assay Buffer (two vials; 43-002)
Beads (premixed)
Biotinylated Reporter Antibodies (premixed)
Streptavidin-PE (one vial; 45-001)
Stop Solution (one vial; 43-001)
Standards (one vial)
96 well filter plate (one plate)
Empty Mixing vials
Flexible kits
Available in 1 plate (100 AP) or 5 plate (500 AP) format
Allow the end user to customise assay specific to his/her
exact requirements
Available for human, mouse and rat
Flexible Assays
The Beadmaster kit
• Multi-Cytokine Standards
• Streptavidin-Phycoerythrin
• Stop Solution
• Assay Buffer
• Mixing Vials
• Detailed Protocols
The Beadmates
• Anti-Cytokine Beads
(22x)
• Anti-Cytokine Reporter
(22x)
Current Beadmates available
MOUSE:
HUMAN:
RAT:
IL-1a
IFN-g
IL-1a
IFN-g
IL-4
IL-1b
IFN-a2
IL-1b
TNF-a
IL-5
IL-2
TNF-a
IL-2
RANTES
GM-CSF
IL-3
TNF-β
IL-3
MIP-1a
TNF-a
IL-4
Eotaxin
IL-4
MIP-1β
IFN-g
IL-5
MCP-1
IL-5
MCP-1
IL-6
RANTES
IL-6
IL-7
MIP-1a
KC
IL-8
MIP-1b
IL-9
IL-9
IP-10
IL-10
IL-10
IL-1ra
IL-12(p40)
IL-12(p40)
sIL-2ra
IL-12(p70)
IL-12(p70)
GM-CSF
IL-13
IL-13
MCP-3
IL-17
IL-15
MDC
GM-CSF
IL-16
TGF-β
Why use Beadlyte Assays over ELISAs?
Parameters
10-plex vs. 10 ELISAs
Time
>Tenfold less hands-on time; similar incubations
Cost of materials
1/3 the cost of ready-made ELISA
Comparable cost to self-made ELISA
Wash steps
Either 0 or 1 compared to 8-12
Sensitivity
Similar
Sample volume
No need to aliquot 10 samples
Can obtain up to 24 cytokines from 25 l compared to 750
l for ELISA
Less sample, more data, less time, less cost!!!!!!
Advantages of Beadlyte Cytokine Assays
Highest Sensitivity (Beadlink) in Optimized Kit
Largest Panel Available (26 and growing)
Cross-Reactivity Data Available
Convenient Cytokine Standards
Minimal Wash Steps
Applications scientists developing alternative protocols
and aiding in custom protocols
Summary
Beadlyte assay range:
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Cytokine/Chemokine/Growth factor detection
Phosphoprotein analysis
Kinase activity assays
Antibody isotyping
For up to date offerings see www.beadlyte.com
Ciaran Sewter [email protected]
07803 951887