1. Beadlyte multiplex assays/Luminex
Ciaran Sewter PhD, Upstate Ltd

2. Presentation Overview
Luminex – the background
Principles of the Luminex Technology
Beadlyte Cytokine Detection Assays

3. 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

4. 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

5. The Use of the Luminex xMap Suspension Array Technology in the Drug Discovery Process
Molecular Biology
Receptor-Ligand
Immunoassay
Enzymatic
Over 75 peer reviewed articles on Luminex technology
Becoming one of the fastest growing technology areas

6. Many Beadlyte® Kits are Bead-Based Sandwich Immunoassays
Bead with capture antibody
Capture antibody binds analyte
Fluorescence labelled reporter antibody binds to capture analyte
Bead ID and reporter quantity determined by laser detector
Laser A
Laser B

7. The flow cell – where it all happens..
Red laser reads the bead, i.e. the target
Green laser detects the amount of the target

8. Principles of the Luminex Technology

9. The Luminex machine

10. Data Capture Software reports results in real-time
Up to 9600 results read in < 1 hour

11. Beadlyte Immunoassay format for the Luminex Machine.
Free excess streptavidin-PE binds to the non-specifically bound biotinylated reporter antibody leading to a signal amplification.
The phycoerythrin is excited by the reporter laser and emits a fluorescence which is quantified by the Luminex reader.
The immune-complex/ microsphere is then excited by the ****** laser. The bead specific emmission is quantified by the luminex and the bead identified.
In assays with high analyte numbers excess biotinylated reporter antibodies bind to the Strep-PE in a non-specific manner.
After another wash step Streptavidin PE is added to the assay. The biotinylated reporter antibody binds to one of the four available sites.
A primary antibody specific for the analyte is
conjugated to the bead surface by an amine coupling reaction
The primary antibody binds to the specific analyte – no crossreactivity with other analytes occurs.
A biotinylated, analyte specific reporter antibody is added to the assay after another wash step.
The Microsphere is a
5.6mM polystyrene bead with two fluorescent dyes incorporated into it in different ratios.

12. 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)

13. Beadlyte – Cytokine / Chemokine Detection
Sample Types
Culture supernatant
Serum
Plasma
Tissue lysates
Urine
Blood spots
Gingival Crevicular Fluid
Nasal Lavage Fluid
Tears
Cerebral Spinal Fluid
Broncho-Alveolar Fluid
Sample Volumes
Routine protocol = 50ul
- s/n = 50ul
- serum = 25ul serum
25ul serum diluent
Have modified low volume protocols
- 25ul, 10ul, 5ul, 1ul

14. 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

15. 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 (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

16. Preconfigured kit components
Assay Buffer (two vials; )
Beads (premixed)
Biotinylated Reporter Antibodies (premixed)
Streptavidin-PE (one vial; )
Stop Solution (one vial; )
Standards (one vial)
96 well filter plate (one plate)
Empty Mixing vials

17. 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

18. Flexible Assays The Beadmaster kit The Beadmates
Multi-Cytokine Standards
Streptavidin-Phycoerythrin
Stop Solution
Assay Buffer
Mixing Vials
Detailed Protocols
The Beadmates
Anti-Cytokine Beads (22x)
Anti-Cytokine Reporter (22x)

19. Current Beadmates available
HUMAN:
IL-1a
IL-1b
IL-2
IL-3
IL-4
IL-5
IL-6
IL-7
IL-8
IL-9
IL-10
IL-12(p40)
IL-12(p70)
IL-13
IL-15
IL-16
IFN-g
IFN-2
TNF-a
TNF-β
Eotaxin
MCP-1
RANTES
MIP-1a
MIP-1b
IP-10
IL-1ra
sIL-2ra
GM-CSF
MCP-3
MDC
TGF-β
MOUSE:
IL-1a
IL-1b
IL-2
IL-3
IL-4
IL-5
IL-6 KC
IL-9
IL-10
IL-12(p40)
IL-12(p70)
IL-13
IL-17
GM-CSF
IFN-g
TNF-a
RANTES
MIP-1a
MIP-1β
MCP-1
RAT:
IL-4
IL-5
GM-CSF
TNF-a
IFN-g

20. 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 ml compared to 750 ml for ELISA
Less sample, more data, less time, less cost!!!!!!

21. 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

22. Summary Beadlyte assay range:
Cytokine/Chemokine/Growth factor detection
Phosphoprotein analysis
Kinase activity assays
Antibody isotyping
For up to date offerings see
Ciaran Sewter