Collaborative Assay Development and Design

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

Research projects that address complex responses need efficient assays

1. The effects of traumatic injuries on the immune system

•  Complex response that involves multiple immune cell types and mediators

•  Mouse and human studies

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 a much information as possible from expensive mouse models and limited amounts of human samples – NIH calls this Sample Sparing Technologies.

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 single 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 for high-throughput studies.

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

Steps to building multiplex assays

1.  Identify target analytes – Done by hypothesis testing and intellectual design.

2. Search for the needed reagents – Ab1, Ab2, standard.

3. Bead coupling optimization – Covalent crosslinking of Ab1 to microplex or magplex bead.

4. Test and optimize assay conditions variables – Screen for detection of standard with Ab2.

5. Validate assay by optimization of assay conditions and sample testing.

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 •  Antibody search using biocompare or labome

websites or trusted suppliers •  Find Abs that have been used in ELISA before •  Look for biotinylated Abs to save time and effort •  Monoclonal on bead, polyclonal as Ab2 is okay

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

Assay development is a step-wise process: Assays developed with Cambridge Biomedical

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0 C X C L 4 te s t c o u p lin g

C o n c e n tra t io n (p g /m l)

MF

I

C X C L 4 S in g le p le x

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0 G a le c tin -9 te s t c o u p lin g

C o n c e n tra t io n (p g /m l)

MF

I

G a le c tin -9 S in g le p le x

Step 1

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

5 0 0 0

1 0 0 0 0

1 5 0 0 0 C X C L 4 1 X v s 2 X A b 1

C o n c e n tra t io n (p g /m l)

MF

I

C X C L 4 2 X A b 1

C X C L 4 1 X A b 1

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

2 0 0 0

4 0 0 0

6 0 0 0 G a le c t in -9 1 X v s 2 X A b 1

C o n c e n tra t io n (p g /m l)

MF

I

G a le c tin -9 2 X A b 1

G a le c tin -9 1 X A b 1

Step 2

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

5 0 0 0

1 0 0 0 0

1 5 0 0 0

C X C L 4 In c u b a t io n b u ffe r v s . s e ru m m a tr ix

C o n c e n tra t io n (p g /m l)

MF

I

S e ru m m a tr ixIn c u b a tio n B u ffe r

1 1 0 0 1 0 0 0 0 1 0 0 0 0 0 00

2 0 0 0

4 0 0 0

6 0 0 0

G a le c t in -9 In c u b a t io n b u ffe r v s . S e ru m m a tr ix

C o n c e n tra t io n (p g /m l)

MF

I

S e ru m m a tr ix

In c u b a tio n B u ffe r

Step 3

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 MIP-2

KC CXCL15

List of validated mouse and human cytokine and other factor assays

Collaborative consumption approach

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.

•  An ongoing need for on-campus Luminex assay development and services – Human Immunology Center, 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 extracts, exhaled breath condensate, etc…

Example: Mouse study to test the influence of mTOR inhibitor treatment on the response to sepsis

IL-1α

0 3 6

Burn/RAPA

Burn

Sham/RAPA

Sham

IL-1β

0 50 100

IL-2

0 15 30

IL-4

0 30 60

IL-5

0 6 12

IL-12p70

0 10 20

*

IL-13

0 6 12

IL-17

0 6 12

*

IL-33

0 10 20

IL-6

080

0016

000

Burn/RAPA

Burn

Sham/RAPA

Sham

IL-10

020

0040

00

*

IL-12p40

030

060

0

IL-18

025

0050

00

IL-23

060

0012

000

IFNγ

010

020

0

*

TNFα

080

016

00

mcp1

080

016

00

GM-CSF

010

020

0

*

Cytokines (pg/mL)

Example: Cytokine Levels in Tear 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

Normal IK Contralateral eye

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

Normal IK Contralateral eye

IL7

Normal IK Contralateral eye

IL8

Normal IK Contralateral eye

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

Normal IK Contralateral eye

MCP1

Normal IK Contralateral eye

IL10

Normal IK Contralateral eye

IL17a

Normal IK Contralateral eye

FGF2

Normal IK Contralateral eye

TREM1

Normal IK Contralateral eye

NS

NS

P=0.04 P=0.01 P=0.02

P=0.004 P=0.02

P=0.003 P=0.01

Ig M

0

2 0 0

4 0 0

6 0 0 ShamS ep s is

MF

I Ig

M

Ig G 1

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

MF

I Ig

G1

Ig G 2 b

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

1 :5 0 1 :4 5 0 1 :1 5 0 0 1 :4 5 0 0

S e ru m D ilu t io n

MF

I Ig

G2

b

Example: 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

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 up opportunities for collaboration.

4.  Developed of VeloceBio as a collaborative biomarker assay development and design company.

5.  Collaboration with Cambridge Biomedical