Using Multiplex Assays and Assay Development to Enhance your Research Program James A. Lederer, PhD Department of Surgery Brigham and Women s Hospital (BWH), BWH Biomedical Research Institute (BWH-BRI) and Harvard Medical School, Boston, MA 2115
Overview Presentation 1. Research interests and reasons for using multiplex assay. 2. Why and how we began using multiplex assays for our research. 3. Luminex assay basics and strategies for developing custom assays. 4. Examples of how we use and take advantage of multiplexing.
Our Research Projects 1. The effects of traumatic injuries on the immune system Complex response that involves multiple immune cell types and mediators Mouse and human studies Systems Immunology 2. Treatments that enhance immunity and restore immune homeostasis after traumatic injuries and severe infections (sepsis). Immuno-monitoring by profiling immune cell phenotypes by flow cytometry and cytokine profiling. In vivo response to injury, infection, and treatments.
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Old school single-mediator assays are good, but multiplexing is more in tune with current scientific needs 1. Difficult to interpret findings and publish results without including more than one mediator or biomarker in dataset. Inflammatory responses and T-cell immune regulation for example 2. More financial demands on research funding requires increasing efficiency and data collection strategies. 3. We need to gather as much information as possible from expensive mouse models and limited amounts of human samples.
History: ELISA to Flow Cytometry Bead Assays to Luminex Assays 1. ELISA basic principle behind all immunoassays Plate-bound antibody antigen antibody sandwiches with color absorbance detection Usually a single analyte approach 2. Flow cytometry-based bead assays Same principle as ELISA but on a bead with a specific detection character for gating on a flow cytometer Limited number of beads can be run as multiplex Not automated or customizable 3. Luminex bead assays Same principle as ELISA using beads with a specific detection character High number of specific beads for multiplexing Customizable and automated
Why do multiplex assays enhance research efficiency? 1. ELISA requires large sample volumes 5 ul for single analyte detection. More samples needed to generate data More reagents needed Need to do multiple wells if more than one analyte If assay does not work, less material for repeat 2. Multiplexing requires much less sample volume for multi-analyte detection. Less sample needed 2 ul or less Less reagents needed Not necessary to do multiple wells Capability to run multiple assays if needed and for future assays from stored samples
Reasons for developing in-house multiplex assays 1. Purchased Luminex instrument in 27 and found discrepancy in results between older technology (BD CBA) and Luminex assays. Several cytokines that were detected by ELISA or CBA were not detected in Luminex assays. 2. Tested several different available kits and found inconsistencies in cytokine detection. Not surprising since assays are likely built with different specific antibody pairs. 3. Some kits detected recombinant cytokines, but not natural cytokines
More reasons for developing in-house Luminex assays 1. Confidence that results will be accurate. 2. Reduce overall costs of performing Luminex assays for our immuno-phenotyping studies. Kit costs were $1-$2/cytokine/96-well plate. Discovered that making in-house assays in bulk would be less expensive. 3. Flexibility for future expansion of panels and for other multiplex assay development opens up capabilities to make a variety of different biomarker assays.
Assay development principles and variables Immuno-assays based on sandwich ELISA principles B Avidin-PE Ab1 Ab2 High specificity due to dependence of signal on bimolecular binding reagents Quality and specificity of assay reaction depends on the nature of Ab1 and Ab2. Antibodies remain the most important component of these and other types of immunoassays
Assay development principles and variables B Avidin-PE Constant Ab1 Ab2 Variables Constant 1. Bead coupling reaction Ab1 Concentration, reaction buffers and chemicals, reaction time, centrifugation washes, blocking buffers 2. Antibody pairing bead-bound (Ab1) versus detection (Ab2), trial and error 3. Detection Ab and Avidin-PE Titration optimization to minimize reagent use and prevent non-specific background 4. Standards - Recombinant versus natural cytokine detection
4,3,2 Luminex bead assay protocol 1. Prepare standard dilutions in buffer similar to sample 2. Prepare mixture of Luminex beads 3. 2 µl of standards or samples added to 96-well plates 4. Bead mix added to plate. 5. Incubate for 4 minutes on rotary shaker. 6. Wash by centrifugation, filter plate, or magnet (MAGPIX). 7. Add mixture of biotinylated-ab2. 8. Incubate 3 minutes. 9. Wash 1. Add streptavidin-pe 11. Incubate for 2 minutes 12. Wash 2X 13. Read on Luminex instrument FlexMap3D, LX2, or MAGPix
Luminex assay principles and readout Ø 5.6 um microsphere are dyed to create distinct colors and can be delineated based on red/infrared content Ø Surface is modified to allow coupling of antibodies, proteins, peptides, or oligonucleotides Ø Immunoassays are based on sandwich ELISA
Data output example: Human IL-4 1. Standards run to calculate sample concentrations 2. Readout is mean fluorescence intensity () 3. Data output from instrument as.csv files that input into MS Excel 4. Plotted to visualize data SampleID Well Location Std1 (.62 ) A1 49 Std2 ( 1.25 ) B1 73 Std3 ( 2.5 ) C1 15 Std4 ( 5. ) D1 175 Std5 ( 1. ) E1 329 Std6 ( 2. ) F1 573 Std7 ( 39. ) G1 127 Std8 ( 78. ) H1 1695 Std9 ( 156. ) A2 2593 Std1 ( 312. ) B2 4439 Std11 ( 625. ) C2 7267 Std12 (125. ) D2 1125 Std13 (25. ) E2 15787 Std14 (5. ) F2 273 Std15 (1. ) G2 22369 Std16 (2. ) H2 22839 1 A3 67 2 B3 1399 3 C3 175 4 D3 129 5 E3 33 6 F3 25 7 G3 1341 8 H3 1447 9 A4 23 1 B4 1411
Recent biomarker development project for lung disease diagnosis COPD vs IPF NIH development project to construct biomarker sets to distinguish patients with chronic obstructive pulmonary disease (COPD) vs. interstitial pulmonary fibrosis (IPF). Ivan Rosas and Fernanda Golzarri, BWH Pulmonary Critical Care and Lovelace Respiratory Research Institute
Steps to Building the COPD/IPF panel 1. Identify target analytes list from literature 2. Search for the needed reagents Ab1, Ab2, standard 3. Find standard Can be difficult for less studied proteins or complex proteins e.g. surfactant protein A (SP-A) 4. micro-batch testing to optimize bead coupling reaction 5. Biotinylation optimization for Ab2, if needed
25 2 15 1 5 Examples: Cytokine Assays In-house mouse cytokine assay Standard Curve Plots Comparing Recombinant Cytokine Detection Assays (Mouse) IL-1β IL-1β Theirs IL-1β Ours 15 1 5 IL-2 IL-2 Theirs IL-2 Ours 15 1 5 IL-4 IL-4 Theirs IL-4 Ours.3125 1 32 124 32768 Concentration of Standard.3125 1 32 124 32768 Concentration of Standard.3125 1 32 124 32768 Concentration of Standard 15 1 5 IL-6 IL-6 Theirs IL-6 Ours 75 5 25 IL-1 IL-1 Theirs IL-1 Ours 125 1 75 5 IL-13 IL-13 Theirs IL-13 Ours 25.3125 1 32 124 32768 Concentration of Standard.3125 1 32 124 32768 Concentration of Standard.3125 1 32 124 32768 Concentration of Standard 125 1 IL-17 IL-17 Theirs IL-17 Ours 35 3 25 TNF TNF Theirs TNF Ours 75 5 25 2 15 1 5.3125 1 32 124 32768 Concentration of Standard.3125 1 32 124 32768 Concentration of Standard
Mouse cytokine assays Detection of Naturally-Produced Mouse Cytokines IL-1β IL-2 IL-4 7.5 Theirs Ours 2 Theirs Ours 25 2 Theirs Ours 5. 2.5 1 15 1 5. Dilution of Supernatant (1:2) Dilution of Supernatant (1:2) Dilution of Supernatant (1:2) IL-6 IL1 IL13 25 2 15 1 5 Dilution of Supernatant (1:2) Theirs Ours 9 8 7 6 5 4 3 2 1 Dilution of Supernatant (1:2) Theirs Ours 2 1 Dilution of Supernatant (1:2) Theirs Ours IL17 TNFα 75 Theirs Ours 125 1 Theirs Ours 5 75 25 5 25 Dilution of Supernatant (1:2) Dilution of Supernatant (1:2)
Human cytokine assay development and testing: Recombinant standards 15 1 5 IL-1a.1 1 1 1 1 1 15 1 5 IL-1b.1 1 1 1 1 1 35 3 25 2 15 1 5 IL-1ra.1 1 1 1 1 1 IL-4 IL-5 IL-6 25 2 1 2 75 15 1 1 5 5 25.1 1 1 1 1 1.1 1 1 1 1 1.1 1 1 1 1 1 9 8 7 6 5 4 3 2 1 IL-7.1 1 1 1 1 1 25 2 15 1 5 IL-8.1 1 1 1 1 1 2 1 IL-1.1 1 1 1 1 1
More recombinant cytokine standard testing IL-12p4 IL-12p7 IL-13 25 7 2 2 6 5 15 1 4 3 1 5 2 1.1 1 1 1 1 1.1 1 1 1 1 1.1 1 1 1 1 1 GM-CSF IFNg TNFb 125 5 7 1 4 6 5 75 5 3 2 4 3 25 1 2 1.1 1 1 1 1 1.1 1 1 1 1 1.1 1 1 1 1 1 TWEAK 25 2 15 1 5.1 1 1 1 1 1
Validate natural cytokine detection: Cytokine production by in vitro stimulated human PBMCs 2 1 natural human cytokine detection None LPS BLP Anti-CD3 2 1 IL-1a Il-1ra IL-1b IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-1 IL-12p4 IL-12p7 natural human cytokine detection part2 2 1 2 None LPS BLP Anti-CD3 1 IL-13 IL-17 IL-21 IL-32 fgf2 g-csf gm-csf IFNg MCP-1 NGF-1 TNFa TNFb TREM-1 TWEAK
Current list of validated mouse and human cytokine and other factor assays Mouse IL-1α IL-1β IL-2 IL-4 IL-5 IL-6 IL-7 IL-1 IL-12(p4) IL-12(p7) IL-23 IL-13 IL-17 IL-18 IL-21 IL-33 IFN-α IFN-γ TNFα G-CSF GM-CSF M-CSF FLT3L SCF MCP-1 Human IL-1α IL-1β IL-1ra IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-1 IL-12/23(p4) IL-12(p7) IL-13 IL-17A IL-18 IL-21 IL-32 FGF-2 G-CSF GM-CSF IFNγ MCP-1 MIP-1α MIP-1β NGF RANTES TNFα TNFβ TREM-1 TWEAK stnf Rl stnf Rll
Collaborative consumption On campus Luminex assay collaborations with reagent replenishment support. A need for on-campus Luminex assay development and services Harvard Catalyst or BWH-BRI Dr. Andrew Lichtman, BWH and HMS mouse models of T cell mediated inflammation Dr. Charles Serhan, BWH and HMS inflammation research in mice and man Dr. Gerry Pier, BWH, Channing Lab vaccine and infectious disease projects Dr. Pedram Hamrah, MGH eye inflammation and infections Dr. Rachel Clark, BWH and HMS human T cell biology in the skin Dr. Arlene Sharpe, HMS mouse models of basic T cell activation mechanisms Dr. Mark Perrella, BWH and HMS mouse stem cells and sepsis responses
Examples of how to use of Luminex multiplexing to increase research efficiency 1. Micro-size experiments to gain more information from less cells 2. Test multiple stimulation conditions 3. Develop more efficient assays to save on reagent costs and sample cost e.g. antibody-isotyping of vaccine assays by multiplex 4. Potential to assay any type of cell or tissue extract e.g. human tears, organ extacts, exhaled breath condensate
Cytokine Levels in Tears Are Correlated with the Corneal Nerve Density and Dendritic Cell Counts in Eyes with Infectious Keratitis Takefumi Yamaguchi, Bernardo Cavalcanti, Pedram Hamrah Mass Eye and Ear Infirmary Shizu Ishikawa, Akinori Osuka, Kentaro Shimizu, James Lederer Department of Surgery, Brigham and Women s Hospital
IL1b IL1Ra IL2 3 P=.2 P=.5 4 NS 25 2 NS 15 15 2 1 5.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye IL6 IL7 IL8 3 P=.4 P <.1 4 P=.3 4 P=.2 15 2 2.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye
IL1 IL17a FGF2 6 P=.1 P=.4 1 P=.2 12 NS 3 5 6.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye GMCSF MCP1 TREM1 2 NS 6 P=.2 P=.4 5 P=.1 P=.3 1 3 25.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye.5 Normal 1.5 IK 2.5 Contralateral 3.5 eye
Multiplexing antigen- or immunogen-specific antibody assays for vaccine testing Immunogen IgM 6 Sham IgM CLP 4 2 B Avidin-PE 15 Ab2 anti-isotype Ab Plasma or serum sample from immunized mouse (or human) IgG1 1 5 IgG1 2 IgG2b 15 1 5 IgG2b 1:5 1:45 1:15 1:45 Serum Dilution
Ovalbumin-peptide induced cytokine production from TCR transgenic T cells, in situ detection with MagPix beads 25 2 15 1 5 TNF CTL sham CTL burn OVA shamova burn OVA 35 3 25 2 15 1 5 IL6 CTL sham CTL burn OVA shamova burn 2 1 IFN-γ CTL sham CTL burn OVA shamova burn 125 1 75 5 25 IL2 CTL sham CTL burnova shamova burn CTL sham CTL burn OVA sham OVA burn 2 IL4 1 IL1 5 IL13 6 IL17 1 75 5 25 4 3 2 1 5 4 3 2 1 CTL sham CTL burn OVA shamova burn CTL sham CTL burn OVA shamova burn CTL sham CTL burn OVA shamova burn CTL sham CTL burn OVA shamova burn
Summary 1. Development of custom multiplex assay is feasible and cost effective through careful optimization and validation. 2. Provides an opportunity to develop new assays that are not commercially available. 3. Opens opportunity for collaboration. 4. Recent development of VeloceBio LLC as a collaborative biomarker development company and potential partnering with Cambridge Biomedical to offer assay development and Luminex assay service.