Using Multiplex Assays and Assay Development to …velocebio.com/attachments/File/website_powerpoint_pdf.pdfUsing Multiplex Assays and Assay Development to Enhance your Research Program

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 02115

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.

From Life Technologies Website

Increased knowledge requires increased data

“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

1. ELISA requires large sample volumes – 50 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 – 20 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

Why do multiplex assays enhance research efficiency?

1. Purchased Luminex instrument in 2007 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

Reasons for developing “in-house” multiplex assays

1.  Confidence that results will be accurate.

2.  Reduce overall costs of performing Luminex assays for our immuno-phenotyping studies.

•  Kit costs were $100-$200/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.

More reasons for developing “in-house” Luminex assays

Assay development principles and variables

Immuno-assays based on sandwich ELISA principles

Ab1 Ab2

B

Avidin-PE

•  High specificity due to dependence of signal on bi-molecular 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

Ab1 Ab2

B

Avidin-PE

Constant Constant Variables

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

Assay development principles and variables

40,30,20 Luminex bead assay protocol 1.  Prepare standard dilutions in buffer similar to sample 2.  Prepare mixture of Luminex beads 3.  20 µL of standards or samples added to 96-well

plates 4.  Bead mix added to plate. 5.  Incubate for 40 minutes on rotary shaker. 6.  Wash by centrifugation, filter plate, or magnet

(MAGPIX). 7.  Add mixture of biotinylated-Ab2. 8.  Incubate 30 minutes. 9.  Wash 10.  Add streptavidin-PE 11.  Incubate for 20 minutes 12.  Wash 2X 13.  Read on Luminex instrument – FlexMap3D, LX200,

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

SampleID Well Location MFI Std01 ( 0.62 pg/ml) A1 49 Std02 ( 1.25 pg/ml) B1 73 Std03 ( 2.50 pg/ml) C1 105 Std04 ( 5.00 pg/ml) D1 175 Std05 ( 10.00 pg/ml) E1 329 Std06 ( 20.00 pg/ml) F1 573 Std07 ( 39.00 pg/ml) G1 1027 Std08 ( 78.00 pg/ml) H1 1695 Std09 ( 156.00 pg/ml) A2 2593 Std10 ( 312.00 pg/ml) B2 4439 Std11 ( 625.00 pg/ml) C2 7267 Std12 (1250.00 pg/ml) D2 11025 Std13 (2500.00 pg/ml) E2 15787 Std14 (5000.00 pg/ml) F2 20073 Std15 (10000.00 pg/ml) G2 22369 Std16 (20000.00 pg/ml) H2 22839

1 A3 67 2 B3 1399 3 C3 175 4 D3 1209 5 E3 33 6 F3 25 7 G3 1341 8 H3 1447 9 A4 23

10 B4 1411

Data output example: Human IL-4

1. Standards run to calculate sample concentrations

2. Readout is mean fluorescence intensity (MFI)

3. Data output from instrument as .CSV files that input into MS Excel

4. Plotted to visualize data

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

Examples: Cytokine Assays – “In-house” mouse cytokine assay

IL-1β

0.03125 1 32 1024 327680

500

1000

1500

2000

2500IL-1β TheirsIL-1β Ours

Concentration of Standard

MFI

IL-2

0.03125 1 32 1024 327680

5000

10000

15000IL-2 TheirsIL-2 Ours


MFI

IL-4

0.03125 1 32 1024 327680

5000

10000



MFI

IL-6

0.03125 1 32 1024 327680

5000

10000



MFI

IL-10

0.03125 1 32 1024 327680

2500

5000



MFI

IL-13

0.03125 1 32 1024 327680

2500

5000

7500

10000



MFI

IL-17

0.03125 1 32 1024 327680

2500

5000

7500

10000



MFI

TNF

0.03125 1 32 1024 327680

500

1000

1500

2000

2500

3000

3500TNF TheirsTNF Ours


MFI

Standard Curve Plots Comparing Recombinant Cytokine Detection Assays (Mouse)

Detection of Naturally-Produced Mouse Cytokines IL-1β

0.0

2.5

5.0

7.5TheirsOurs

Dilution of Supernatant (1:2)

MFI

IL-2

0

10000

20000TheirsOurs


MFI

IL-4

0

50

100

150

200

250TheirsOurs


MFI

IL-6

0

500

1000

1500

2000

2500TheirsOurs


MFI

IL10

0100200300400500600700800900

TheirsOurs


MFI

IL13

0

1000

2000TheirsOurs


MFI

IL17

0

250

500

750TheirsOurs


MFI

TNFα

0

250

500

750

1000

1250TheirsOurs


MFI

Mouse cytokine assays

Human cytokine assay development and testing: Recombinant standards

IL-1a

0.1 1 10 100 1000 100000

5000

10000

15000

pg/ml

MFI

IL-1b

0.1 1 10 100 1000 100000

5000

10000

15000

pg/ml

MFI

IL-1ra

0.1 1 10 100 1000 100000

500

1000

1500

2000

2500

3000

3500

pg/ml

MFI

IL-4

0.1 1 10 100 1000 100000

5000

10000

15000

20000

25000

pg/ml

MFI

IL-5

0.1 1 10 100 1000 100000

10000

20000

pg/ml

MFI

IL-6

0.1 1 10 100 1000 100000

2500

5000

7500

10000

pg/ml

MFI

IL-7

0.1 1 10 100 1000 100000

100020003000400050006000700080009000

pg/ml

MFI

IL-8

0.1 1 10 100 1000 100000

5000

10000

15000

20000

25000

pg/ml

MFI

IL-10

0.1 1 10 100 1000 100000

10000

20000

pg/ml

MFI

IL-12p40

0.1 1 10 100 1000 100000

500

1000

1500

2000

2500

pg/ml

MFI

IL-12p70

0.1 1 10 100 1000 100000

1000

2000

3000

4000

5000

6000

7000

pg/ml

MFI

IL-13

0.1 1 10 100 1000 100000

1000

2000

pg/ml

MFI

GM-CSF

0.1 1 10 100 1000 100000

2500

5000

7500

10000

12500

pg/ml

MFI

IFNg

0.1 1 10 100 1000 100000

1000

2000

3000

4000

5000

pg/ml

MFI

TNFb

0.1 1 10 100 1000 100000

1000

2000

3000

4000

5000

6000

7000

pg/ml

MFI

TWEAK

0.1 1 10 100 1000 100000

500

1000

1500

2000

2500

pg/ml

MFI

More recombinant cytokine standard testing

Validate natural cytokine detection: Cytokine production by in vitro stimulated human PBMCs

natural human cytokine detectionIL

-1a

Il-1r

a

IL-1

b

IL-2

IL-3

IL-4

IL-5

IL-6

IL-7

IL-8

IL-9

IL-1

0

IL-1

2p40

IL-1

2p70

0

100

200

NoneLPSBLPAnti-CD3

10000

20000

MFI

natural human cytokine detection part2

IL-1

3

IL-1

7

IL-2

1

IL-3

2

fgf2

g-C

SF

gm-C

SF

IFN

g

MC

P-1

NG

F-1

TNFa

TNFb

TREM

-1

TWEA

K0

100

200

10000

20000 NoneLPSBLPAnti-CD3

MFI

Mouse IL-1α IL-1β IL-2 IL-4 IL-5 IL-6 IL-7 IL-10

IL-12(p40) IL-12(p70)

IL-23 IL-13 IL-17

Human IL-1α IL-1β IL-1ra IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10

IL-12/23(p40) IL-12(p70)

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

IL-18 IL-21 IL-33 IFN-α IFN-γ TNFα G-CSF

GM-CSF M-CSF FLT3L SCF

MCP-1

Current list of validated mouse and human cytokine and other factor assays

Collaborative consumption

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

•  On campus Luminex assay collaborations with reagent replenishment support.

•  A need for on-campus Luminex assay development and services – Harvard Catalyst or BWH-BRI

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

0

5000

10000

15000

20000

25000

0.5 1.5 2.5 3.5 0

1500

3000

0.5 1.5 2.5 3.5

IL1b

Normal IK Contralateral eye 0

200

400

0.5 1.5 2.5 3.5

IL1Ra

Normal IK Contralateral eye

IL2


0

15000

30000

0.5 1.5 2.5 3.5 0

200

400

0.5 1.5 2.5 3.5 0

2000

4000

0.5 1.5 2.5 3.5

IL6


IL7


IL8


P=0.005 P=0.02

P < 0.001 P=0.04

P=0.002 P=0.03

NS NS

0

300

600

0.5 1.5 2.5 3.5 0

5000

10000

0.5 1.5 2.5 3.5 0

6000

12000

0.5 1.5 2.5 3.5

0

1000

2000

0.5 1.5 2.5 3.5 0

3000

6000

0.5 1.5 2.5 3.5 0

2500

5000

0.5 1.5 2.5 3.5

GMCSF


MCP1


IL10


IL17a


FGF2


TREM1


NS

NS

P=0.04 P=0.01 P=0.02

P=0.004 P=0.02

P=0.003 P=0.01

IgM

0

200

400

600 ShamCLP

MFI

IgM

IgG1

0

5000

10000

15000

MFI

IgG

1IgG2b

0

500

1000

1500

2000

1:50 1:450 1:1500 1:4500

Serum Dilution

MFI

IgG

2b

Multiplexing antigen- or immunogen-specific antibody assays for vaccine testing

Ab2 – anti-isotype Ab

B

Avidin-PE

Plasma or serum sample from immunized mouse (or human)

Immunogen

Ovalbumin-peptide induced cytokine production from TCR transgenic T cells, in situ detection with

MagPix beads IFN-γ

CTL sham CTL burn OVA shamOVA burn0

1000

2000

TNF


50

100

150

200

250IL6


500

1000

1500

2000

2500

3000

3500IL2

CTL sham CTL burnOVA shamOVA burn0

2500

5000

7500

10000

12500CTL shamCTL burnOVA shamOVA burn

IL4


100

200IL10


250

500

750

1000

IL13


100

200

300

400

500IL17


1000

2000

3000

4000

5000

6000

OVA

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.

Documents

Using Multiplex Assays and Assay Development to …velocebio.com/attachments/File/website_powerpoint_pdf.pdfUsing Multiplex Assays and Assay Development to Enhance your Research Program