Multiplexing as Essential Tool for Modern Biology

Transcription

1 Multiplexing as Essential Tool for Modern Biology Bio-Plex Seminar, Debrecen, Gyula Csanádi, PhD.

2 The "Age of "-omics" Studying interrelationships at different level of complexity Genes - Unveiling genetic information SNP scoring, allelic discrimination Cell - Following intracellular processes Gene expression studies Cellular signaling Proteome analysis Organism - Tracing physiological conditions Cytokine profiling Autoimmune & allergy processes Clinical diagnosis

3 How can Multiplexing Methods support Systems Biology? Benefits of Multiplexing " A multiplex assay is a type of laboratory procedure that simultaneously measures multiple analytes (dozens or more) in a single assay. " Increase amount of information from a single sample Decrease volume requirements for precious samples (tissue samples, tumor biopsies, newborn screening) Quantify analytes and interrelationships between analytes with higher correlations (in a single sample) Data reliability: same instrument, same operator, same day, single run Reduce reagent, expense and labor

4 How to Multiplex? Several technologies available DNA, Protein Chips Real-Time PCR 2-D Arrays 2-D Electrophoresis Parameters to consider Sample type: DNA, RNA vs. proteins Multiplexing capacity Quantification dynamic range Throughput

5 How to Multiplex? Microarrays RT- qrt-pcr 2-D arrays DNA, protein chips High data throughput(whole genome, differencial proteome) Incredible dynamic range (10 orders) Correlation coefficient 0.99 Limited multiplexing capacity: 4-6x Limited dynamic range (2 3 orders), data needs to be verified by other, methods Limited sample throughput High throughput / speed run completed within hrs

6 How to the Protein level: 2-D electrophoresis certain proteins moderate reproducibility Dynamic range 3 5 orders (staining technology) Low throughput Mass Spectrometry Low dynamic range (2-3 orders) Middle multiplexing capacity: 1 order High throughput 6

7 What to the Protein level: ELISA method Correlation coefficient 0.99 Limited multiplexing capacity: 1-4x Low throughput Western blotting method

8 How to Multiplex ELISA & Western Methods? xmap Bead-based technology: Assay occurs on bead surface µm polystyrene bead diameter Multiplexing enabled by bead color Beads with different ratios of red and infrared dyes Up to 100 uniquely colored beads

9 Positioning of xmap multiplexing xmap Protein microarrays and proteomics Gavin MacBeath, Nature Genetics 32, (2002)

10 Positioning of xmap multiplexing per Sample microarray array-of-arrays Tests multiplex RT-PCR Mass Spec 3D, xmap Technology northern blot Real-Time PCR Sample throughput

11 How to Multiplex? Suspension arrays (xmap - multi analyte profiling) Sandwich immunoassay Nucleic Acid Good dynamic range 3.5 orders Sufficient multiplex capacity up to 100, high sample throughput CV < 8% Many applicable formats Ligand binding Enzymatic FI 30,000 25,000 20,000 15,000 10,000 5,000 0 Mouse cytokine 8-Plex Assay ,000 10, ,000 Log pg/ml IL-b TNFa IL-4 IFNg IL-2 IL-5 IL-10 GMCSF xmap

12 How to Multiplex? The xmap Technology xmap ~6 µm microspheres SEM photo of 3.4 micron particles on human hair, 1000x

13 How to Multiplex? The xmap Technology xmap Multiplexing enabled by bead color Dual coated beads with different ratios of red and infrared dye 10 concentrations of each dye Up to 100 uniquely colored beads Red (660 nm) IR(infrared) (730 nm)

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17 How to Multiplex? The xmap Technology xmap Technology: 100 distinctly colored microsphere sets We use a combination of two dyes to distinguish one bead set from another Each bead set exhibits a unique fluorescence of red and infrared dye All beads that have a particular color ratio belong to a specific bead set which is identified with a unique bead ID number

18 How to Multiplex? The xmap Technology Each bead set has a predetermined location on the bead map

19 How to Multiplex? The xmap Technology 100 different tests and be measured in a single reaction volume Different microsphere sets may be combined Even when combined, each bead set retains its signature

20 How to Multiplex? The xmap Technology Bead Address Establishes Analyte for Instrument IL-9 IL-6 xmap Each bead type is assigned an analyte TNFα IL-1β 77 92

21 Multiplexing with Colored Bead Sets Antibodies are immobilized on 5 micron beads instead of microplate well

22 Multiplexing with Colored Bead Sets The antibody-conjugated beads are mixed into a single suspension. Here we have a 4-plex

23 The bead suspension is mixed with biological sample such as serum, plasma, cell lysate, or tissue culture media for detection of multiple analytes at once.

24 Suspension Bead Array Technology IL-6 IL-9 xmap IL-1b TNF-a *SA-PE *Streptavidin-Phycoerythrin

25 How to Multiplex? The xmap Technology xmap

26 How to Multiplex? The xmap Technology Dual Laser Excitation xmap

27 How to Multiplex? The xmap Technology Beads on a String

28 How to Multiplex? The xmap Technology Sample Injection Point Bead Interrogation Sheath fluid Classification 1 Classification 2 Doublet Discriminator Red Laser Signal Quantification

29 How to Multiplex? The xmap Technology Histogram - Doublet Discriminator: Light Scatter - Bead Size Intensity Plot - Bead Fluorescence: Ratio Cl1 / Cl2- Analyte Identification Debris Single beads Aggregated beads Histogram - displays the # of events vs. signal The height of the signal in each channel represents the # events

30 How to Multiplex? The xmap Technology 100 color code regions Each bead maps to a specific region of the array based on the ratio of the two classification signals

31 PMT detector integration PMT signal strength is proportional to concentration xmap Mouse cytokine 8-Plex Assay FI 30,000 25,000 20,000 15,000 10,000 5, ,000 10, ,000 Log pg/ml IL-b TNFa IL-4 IFNg IL-2 IL-5 IL-10 GMCSF

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