BioPlexTechnology: an overview of hardware,...

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: LSG_TechServ_US@bio-rad.com

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?