BioPlex Technology: an overview of hardware, software, maintenance and data analysis.

Transcription

1 BioPlex Technology: an overview of hardware, software, maintenance and data analysis. Alasdair I. Reid Field Application Specialist Bio-Rad Laboratories

2 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

3 Bio-Rad Laboratories life science research & diagnostics Driving innovations: Chromatography Solutions Electrophoresis Technology Proteomics and Imaging Systems Multiplex Protein Assays Protein Interaction Technology

4 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

5 Protein Analysis single vs multiplex Protein microarrays and proteomics Gavin MacBeath, Nature Genetics 32, (2002)

6 Assay Technologies from Tubes to Bead Assays Technology platform basics Industry advances in multiplex and parallel analysis Test tubes Microwell plate Microarray Bead Technology: Microsphere-based liquid array

7 Why Multiplex? 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)

8 xmap Technology Applications 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

9 Bio-Plex: A Flexible Multiplexing Platform 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.

10 12 years of Luminex Leadership 2010 BPM 6.0 launched

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12 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

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15 Suspension Bead Array Technology IL-6 IL-9 Multiplexing is Achieved with sets of Microspheres Encoded with a Unique Spectral Address TNFa IL-1b

16 Suspension Bead Array Technology

17 Bead and Analyte Identification Histogram - Doublet Discriminator: Light Scatter - Bead Size Intensity Plot - Bead Fluorescence: Ratio Cl1 / Cl2- Analyte Identification

18 Bio-Rad Bio-Plex and Bio-Plex Manager 6.0 Software

19 Bio-Plex Manager 6.0 Software and MCV Plate 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

20 MCV Plate IV and Reservoir Automated setup, run, shutdown, calibration and validation functions.

21 The Quick Guide follow the steps Bio-Plex Manager Software

22 Simple Calibration Protocol

23 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!!!

24 Validation - MONTHLY

25 Simple Validation Protocol

26 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) Validation Quantitative output suitable for IQ/OQ use Performed monthly, during troubleshooting or if system is moved

27 Bio-Plex Manager Software Step 1 Author s Name Experimental Notes

28 Bio-Plex Manager Software Step 2 Select Panel Add Panel Edit Panel Transfer Analytes to Selected List

29 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)

30 Bio-Plex Manager Software Step 3 Assign: B = Blank C = Control S = Standard X = Sample Right Click Wells to Set Identifier Names Use Plate Grouping to set Reference Set for Phospho Assays

31 Run Multiple Experiments and one plate!!!

32 Bio-Plex Manager Software Step 4 Enter S1 Concentration for each Analyte / Dilution Factor / Press Calculate Save Lots Reuse Lots Import Stds

33 Enter Concentration for each Control Bio-Plex Manager Software Step 5

34 Bio-Plex Manager Software Step 6 Enter Sample Identifiers Or Right Click on Plate Map Enter dilution factors

35 Bio-Plex Manager Software Step 7 Add Bead Count Select High RP1 to increase signal from PMT Start the Run Save Reservoir functions: add pre/post run steps Advanced Options Set Volume Pauses

36 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.

37 Data Acquisition View and Raw Data in rbx. file Event Histogram Bead Map Median FI (sample size) Bead Statistics Sampling Errors

38 Report Table assess assay performance Sensitivity Precision Accuracy Conc in Range

39 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

40 Fluorescence Intensity (FI) Defining the working range of the assay Typical calibration curve for IL-10, level of quality controls, definition of working range ULOQ Working range LLOQ Quality control Concentration (pg/ml)

41 Standard Curve Tab Curve Fit Optimizer defines the working range of the assay for you!!

42 Optimization Controls Feedback on currently selected curve (or all if all optimized) Optimize Curve(s) Apply actions to all curves Undo last optimization (and return to previous state) Clear any outliers in curve(s)

43 Bio-Plex Manager 6.0 Software Analyses the Data Sophisticated Curve Fitting and Statistical Tools (StatLea from Brendan Scientific) Unknowns and Controls

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45 Graphing Features Create new graphs with specific samples / anlaytes Right Click > Copy > Paste

46 Integrated Online Help

47 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

48 Sample Types Human Tissue Culture Supernatant Serum Plasma Adipose Interstitial Fluid Bronchoalveolar Lavage Fluid Cerebrospinal Fluid Nasal Lavage Fluid Peritoneal Fluid Synovial Fluid Rat Tissue Culture Supernatant Serum Plasma Bronchoalveolar Lavage Fluid Nervouse System Tissue Colon Tissue Bulletin 5297 Mouse Tissue Culture Supernatant Serum Plasma Bronchoalveolar Lavage Fluid Synovial Fluid/Patellar Washouts Colon Tissue Kidney Tissue Lung Tissue Nervouse System Tissue Spleen Tissue Misc Urine Blister Fluid Eyeball Jelly Tears Breath Condensates Dried Bloodspots Breast milk Skin Tissue

49 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

50 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

51 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

52 Cytokine Assays

53 Cytokine assay workflow 1. Mix antibody coated beads with samples, standards and controls 2. Add specific biotinylated detection antibodies 30 mins incubation 4. Read assays in Bio-Plex 200 Wash between each step 30 mins incubation 3. Detection: streptavidin-pe 10 mins incubation Under 3 hours start to finish

54 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)

55 Bio-Plex Assay Menu Total: 91 Cytokine, Chemokine and Growth Factors Assays 50 Human Assays 33 Mouse Assays Available as x-plex Assays and Express

56 Disease Panels

57 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: BIORAD ( ), option #2 Online technical support and worldwide contact information are also available at consult.bio-rad.com.

58 Serial # for Tech Support 1. Click! 2. Go to Version 3. Record Serial #

59 Thank you Any Questions?