BioPlex Technology: an overview of hardware, software, maintenance and data analysis.
Alasdair I. ReidField Application Specialist
Bio-Rad Laboratories
Content
•BioPlex Technology: an overview of hardware, software, maintenance and data analysis.
Alasdair I. Reid; Field Application Specialist, Bio-Rad Laboratories
• Introduction to Bio-Rad
• Understanding Protein Networks
• Why and How of Multiplexing
• xMAP technology
• Bio-Plex hardware and the MCV Plate
• BioPlex software – from Protocols to Data Analysis
• Sample Types and the BioPlex Plate Washer
• Bio-Rad’s products for Cytokine research
• Contacting Bio-Rad Tech Support
Bio-Rad Laboratories
life science research & diagnostics
Driving innovations:
▫ Chromatography Solutions
▫ Electrophoresis Technology
▫ Proteomics and Imaging Systems
▫ Multiplex Protein Assays
▫ Protein Interaction Technology
Functional Protein Analysis –Understanding Protein Networks
• Cell Physiology
• Cell-to-Cell Interaction
• Influence of External Factors
•Need to understand gene function & protein interaction
•Need to understand cell signaling
•Need to increase efficiency in research and drug discovery
•Need to increase throughput in proteomics research
Protein microarrays and proteomicsGavin MacBeath, Nature Genetics 32, 526 – 532 (2002)
Protein Analysis single vs multiplex
Assay Technologiesfrom Tubes to Bead Assays
• Test tubes • Microwell plate • Microarray • Bead Technology:Microsphere-based
liquid array
•Technology platform basics
–Industry advances in multiplex and parallel analysis
•Decrease precious sample volume requirements
•Reduce assay reagent volume, expense and labor
•Include assay controls in each well, rather than on each plate
•Dramatically increase the amount of information from a single sample
•Better correlation of data between different targets (target analysis is simultaneous for each sample)
Why Multiplex?
Life Science Research
• Extracellular signaling proteins
• Intracellular signaling proteins
• Cancer markers
• Cardiac markers
• Endocrine
• Isotyping
• Metabolic Markers
• Neurobiology
• Transcription Factors
• Gene Expression
• Genotyping
• MicroRNA profiling
Diagnostics
• Genetic Disease
• Allergy Testing
• Autoimmune Disease
• HLA Testing
• Infectious Disease
• Biodefense
xMAP Technology Applications
•Integrated, easy-to-use 96-well plate-based suspension analysis system.
•Simultaneous quantitative analysis of up to 100 different proteins, peptides,
DNA fragments and RNA fragments from a single drop of sample.
•Novel bead-based technology licensed from Luminex Corporation.
•Often referred to as xMAP technology.
Bio-Plex: A Flexible
Multiplexing Platform
12 years of Luminex Leadership
2010 – BPM 6.0 launched
Suspension Bead Array Technology—How is
Multiplexing Accomplished?
Bead-based technology:
•Licensed for Luminex Corporation (Austin, TX.)
•Assay occurs on bead surface
• 5.6µm or 6.5µm or 8.0um bead diameter
• Multiplexing enabled by bead “color”
• Beads with different ratios of red and infrared dyes
• Up to 100 uniquely colored beads
Suspension Bead Array Technology
Multiplexing is Achieved
with sets of
Microspheres Encoded
with a Unique Spectral
Address
IL-9
IL-6
TNFa
IL-1b
Suspension Bead Array Technology
Bead and Analyte Identification
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Histogram -Doublet Discriminator:Light Scatter - Bead Size
Intensity Plot -Bead Fluorescence:Ratio Cl1 / Cl2- Analyte Identification
Bio-Rad Bio-Plex and Bio-Plex
Manager 6.0 Software
•Automated setup, run, and shutdown functions
•Automated calibration and validation functions
•Novel MCV Plate
•Simple and flexible multiplex template formatting
•Automated system monitoring and error flags
•Automated generation of multiple standard curves and stats
•Sorting and screening tools
Bio-Plex Manager 6.0™ Software and MCV Plate
MCV Plate IV and
Reservoir
Automated setup, run, shutdown,
calibration and validation
functions.
Bio-Plex Manager Software
The Quick Guide – follow the steps
Simple Calibration Protocol
Calibration - DAILY
Standardization of daily signal output to a known fluorescence signal
• Uses specific calibration beads with a stable fluorescence signal
• Adjusts detector voltages to this standard signal
• Also used to monitor laser life, detector function,and optical alignment
Pass / Fail!!!
Validation - MONTHLY
Simple Validation Protocol
• Verification that the Bio-Plex system is operating within
defined specifications with respect to:
Optical alignment
Reporter channel performance
Classification efficiency
Integrity of fluidics (carryover)
• Quantitative output suitable for IQ/OQ use
• Performed monthly, during troubleshooting or if system is moved
Validation
Bio-Plex Manager Software – Step 1
Author’s
Name
Experimental
Notes
Bio-Plex Manager Software – Step 2
Select Panel
Add Panel
Edit Panel
Transfer Analytes to ―Selected List‖
Bio-Plex Manager Software – Step 2a
To Add a
Panel
Name Panel
Select xMAP
bead type
Select Add
Assign Bead
Region (i.e.
58) and Name
(i.e. IL-2)
Bio-Plex Manager Software – Step 3
Assign:
B = Blank
C = Control
S = Standard
X = Sample
Use Plate Grouping to set Reference Set for Phospho Assays
Right Click
Wells to Set
Identifier Names
Run Multiple Experiments and one plate!!!
Bio-Plex Manager Software – Step 4
Enter S1 Concentration for each Analyte / Dilution Factor / Press Calculate
Save Lots
Reuse Lots
Import Stds
Bio-Plex Manager Software – Step 5
Enter Concentration for each Control
Bio-Plex Manager Software – Step 6
Enter
Sample
Identifiers
Or Right
Click on
Plate Map
Enter
dilution
factors
Bio-Plex Manager Software – Step 7
Add Bead Count
Reservoir
functions: add
pre/post run
steps
Advanced
Options
Set Volume
Pauses
Select High RP1 to increase signal from PMT
Start the Run Save
Bio-Plex Reservoir
The reservior fits in to the XY platform to the right of
the MCV plate
Automated maintenance/walk-away feature setting up and
automatically running unattended ―pre‖ or ―post‖ run washes, as
well as shut-down operations.
Sampling Errors
Event HistogramBead Map
Median FI (sample size) Bead Statistics
Data Acquisition View
and Raw Data in rbx. file
Report Table – assess assay performance
Sensitivity
Precision
Accuracy
Conc in
Range
Assay Performance Parameters
Sensitivity
•Limit of detections (LOD, background + 2 SD)
Precision
•Coefficient of variance (%CV, SD/mean)
•Intra-assay
•Inter-assay
Accuracy
•Recovery (observed conc/expected conc)
•Standards
•Spiked controls
•Spiked samples
•Experimental error•Pipetting errors
•Deviations from assay protocols
•Reconstitution times
•Incubation time
•Inconsistent time management
•Sample handling errors
•Freeze thaw cycles
•Variations in storage conditions
•Sample Matrix effects
Defining the working range of the assay
•Typical calibration curve for IL-10, level of quality controls, definition of working range
10.00 100.00 1000.00 10000.00
Concentration (pg/mL)
0.00
10000.00
20000.00
Flu
ore
scence Inte
nsity (
FI)
Quality controlLLOQ
ULOQ
Working range
Curve Fit Optimizer – defines the working range of the assay for you!!
Standard Curve Tab
Optimization Controls
Apply
actions to all
curves
Optimize
Curve(s)
Undo last
optimization
(and return to
previous state)
Clear any
outliers in
curve(s)
Feedback on currently selected curve (or all if all optimized)
Bio-Plex Manager 6.0 Software Analyses the Data
Sophisticated Curve Fitting and Statistical Tools (StatLea from Brendan Scientific)
Unknowns
and
Controls
Graphing Features
Create new
graphs with
specific
samples /
anlaytes
Right Click >
Copy > Paste
Integrated Online Help
Research Areas
• Cancer
• Stroke
• Dialysis (peritonitis)
• Β-Amyloid
• Intoxication
• Cerebral Infarction
• Reumatoid Arthritis
• Hepatitis C
• Thromboembolism
• Kashin Beck Syndrome
• Lupus Nephritis
• PolyCystic Kidney Disease
• Preeclampsia
• Preterm Labour
• Respiratory Diseases
• Endometriosis
• Marijuana Users
• Obese Patients
• Myocardial Infarction
• Calcium Oxalate Stones
• HIV
• Cystic Fibrosis
• Cardiovascular Disease
• Schizoprenia
• Arsenic Exposure
• Allograft Rejection
• Gaucher’s Disease
• Diabetes
• Parasitic Infections
• Osteoporosis
• Hypertension
• Chron’s Disease
Sample Types
Bulletin 5297
Human
•Tissue Culture Supernatant
•Serum
•Plasma
•Adipose Interstitial Fluid
•Bronchoalveolar Lavage Fluid
•Cerebrospinal Fluid
•Nasal Lavage Fluid
•Peritoneal Fluid
•Synovial Fluid
Mouse
•Tissue Culture Supernatant
•Serum
•Plasma
•Bronchoalveolar Lavage Fluid
•Synovial Fluid/Patellar Washouts
•Colon Tissue
•Kidney Tissue
•Lung Tissue
•Nervouse System Tissue
•Spleen Tissue
Rat
•Tissue Culture Supernatant
•Serum
•Plasma
•Bronchoalveolar Lavage Fluid
•Nervouse System Tissue
•Colon Tissue
Misc
•Urine
•Blister Fluid
•Eyeball Jelly
•Tears
•Breath Condensates
•Dried Bloodspots
•Breast milk
•Skin Tissue
Bio-Rad’s Cytokine assays are on Magnetic Beads
•Compared to polystyrene bead based assays magnetic bead-based assays offer:
•Freedom from the vacuum plate
•Data consistency
• Improved workflow
• Increases Sample repertoire
With Magnetic Beads you can use the automated Plate Washer
•Magnetic (Bio-Plex Pro)
–Magnetic plate carrier
–Standard ELISA carrier
•Magnetic and Vacuum (Bio-Plex Pro II)
–Vacuum plate carrier and magnetic plate carrier
–Runs both polystyrene or magnetic bead-based assays
Vacuum Manifold vs. Bio-Plex Pro Wash Station
Vacuum Manifold Approach
1. Place plate on vacuum manifold
2. Apply vacuum and set vacuum strength
3. Filter plate
4. Remove plate, blot dry
5. Add buffer using multichannel pipet
6. Repeat steps 3 through 5 (wash 2)
7. Repeat steps 3 though 5 (wash 3)
8. Add next reagent to plate
Vacuum Facts
• Total time to complete varies with user
• Results can vary with experience of user
• Consistent results plate to plate is a challenge
Bio-Plex Pro Approach
1. Place assay plate on Bio-Plex Pro
2. Select wash program, press start
3. Add next reagent to plate
Bio-Plex Pro Facts
• Total time to complete wash step 3-4 minutes
• Equivalent or better than manual manifold
• Expert consistency in novice’s hands
Cytokine Assays
Cytokine assay workflow
1. Mix antibody coated beads with samples, standards and controls
3. Detection:
streptavidin-PE
30 mins
incubation
2. Add specific
biotinylated
detection
antibodies
30 mins
incubation
10 mins incubation
4. Read assays in Bio-Plex 200
Under 3 hours start to finish
Wash between
each step
Bio-Plex Pro Cytokine Assays -MAGPLEX
•All-in-one kit format
–Assay buffer
–Wash buffer
–Detection antibody diluent
–Streptavidin-PE
–Sealing tape
–96-well plate (filter plate)
–Universal standard diluent
–Universal sample diluent
–Instruction manual
•Same all-in-one kit (human, mouse)
Total: 91 Cytokine, Chemokine and Growth Factors Assays
50 Human Assays
33 Mouse Assays
Available as
x-plex Assays
and Express
Bio-Plex Assay Menu
Disease Panels
Technical Support
Bio-Rad Life Science Group Technical Support
Technical support is available Monday through Friday,
from 7:00 AM to 7:00 PM, Central Time.
Phone: 1-800-4BIORAD (1-800-424-6723), option #2
Email: [email protected]
Online technical support and worldwide contact information are also
available at consult.bio-rad.com.
Serial # for Tech Support
1. Click !
2. Go to Version
3. Record Serial #
Thank you
Any Questions?