Development of fully automated ultrasensitive digital immunoassays using Single Molecule Array

(Simoa™) technologyKhushbu Bhatt, Andrew Hebert, Mikhail Levit

Sanofi Oncology, 640 Memorial Drive, Cambridge, MA 02138

Abstract Purpose: Accurate analysis of protein biomarkers and cytokines is of

utmost importance for determining the target expression, therapeutic

efficacy, and toxicity in anti-cancer immunotherapeutics. However,

currently available immunoassays on ELISA and Meso Scale Discovery

platform are often not sensitive enough to detect low levels of proteins.

Method: Quanterix’s Simoa (Single molecule array) is highly sensitive,

fully automated immunoassay technology which makes use of arrays of

femtoliter sized chambers to isolate and detect single immuno-complex

on paramagnetic beads. This leads to detection of proteins in

femtomolar concentrations thus improving the sensitivity over

traditional immunoassays. It utilizes coupling of capture antibodies to

the paramagnetic beads followed by addition of analyte and

biotinylated antibodies which are labelled with the enzyme

streptavidin-β-galactosidase (SβG) and resorufin-β-galactopyranoside

as the substrate.

Result: Simoa based cytokine immunoassays were developed and

optimized to determine the target expression in murine model of tumor.

Variables like capture and detection antibody, SβG concentration, bead

number, diluent formulations, number of steps in the assay and the

incubation time were optimized. Assays with 10-200 fold improvement

in the sensitivity over Meso Scale Discovery and ELISA were developed

and validated.

Introduction: Simoa technology

Simoa platform makes use of sandwich immunoassay format

comprising of 3 steps:

1. Capture of analyte on the paramagnetic beads coated with the capture

antibody

2. Addition of biotinylated detection antibody to form an immuno-

sandwich

3. Labelling the detection antibody with the enzyme streptavidin-β-

galactosidase (SβG)

The instrument carries out three washings between each of these steps

Simoa assay can be carried out either in 3-step or 2-step or 1-step

format

1. 2-step assay- Incubating the detector with the analyte and the capture

beads prior to washing

2. 1-step assay- Incubation of all the components together

The capture beads with the immunocomplexes are resuspended in RGP

(resorufin β-galactopyranoside) substrate.

Beads are then pulled into an array of 216,000 fL sized wells, which

holds no more than single beads per well and the array is sealed by oil.

RGP substrate is hydrolyzed by SβG into fluorescent molecule

resorufin in the microwell if the analyte is captured and labelled.

Each single bead molecule generates high fluorescence signal locally

enabling digital readout and permitting measurements at femtomolar

concentrations.

Results

+

Results

Conclusion

Quanterix’s Simoa™ platform offers higher sensitivity and automation

for efficient processing of the samples with significant reduction in the

assay development time.

Simoa based cytokine immunoassays were developed and validated for

analyzing the concentration of cytokine in mice serum and tumor

models to ascertain the target expression

One to two orders of magnitude improvement in sensitivity was

achieved as compared to ELISA and Meso Scale Discovery

Assays were optimized for low background, high signal-to-noise ratio

and low matrix effect

Assays were evaluated for sensitivity, dilution, spike and recovery in

serum and tumor lysates.

Previously undetectable levels of the cytokine were detected in serum

and tumor lysates of the groups treated with the mRNA based

immunotherapy using the immunoassays developed on Simoa

platform.

Acknowledgement

I would like to thank my mentor Mikhail Levit and my team member

Andrew Hebert for training me in the lab and helping me with the

project.

Thanks to Timothy Wagenaar, Patrick Guirnalda, Tatiana Tolstykh,

Yu-an Zhang, Michael Lampa for assisting with the assay development

and optimization.

I would also like to thank the Biochemistry and Bioanalytics

department head Hong Cheng for her continued motivation, guidance,

support and patience during my time in Sanofi.

Assay development outline

Capture ab

Biotinylated detection ab

SβG

Chimeric protein assay development

Assay 3-step

Beads/reaction 500,000

Detector (ug/ml) 0.3

SβG (pM) 150

1. Determine the capture and detection ab

Capture ab: Y, detection ab: X

Higher background: 0.078 AEBs (acceptable < 0.02)

LLOD: 0.6 pg/ml

2. Optimize reagent concentration

Assay 2-step

Beads/reaction 250,000

Detector (ug/ml) 0.15

SβG (pM) 25

Chimeric Protein (X-Y)

Increased slope

Reduced background: 0.0078 AEBs

LLOD: 0.05 pg/ml, Range: 0.05-200 pg/ml

3. Simoa v/s Meso Scale Discovery (MSD)

2.5 log improvement in sensitivity

4. Analyze protein expression in B16F10 tumor model

Chimeric Protein (X-Y)

Expression in Tumor Model

G P N 7 5 5 0

pg

/mg

to

ta

l p

ro

te

in (

lys

ate

)

M ix 1 M ix 2 M ix 3 M ix 4

-1 0 0

0

1 0 0

2 0 0

3 0 0

4 0 0

m R N A m ix tu re s e n c o d in g im m u n o -th e ra p e u tic p ro te in s

Target expression was possible in the tumor lysates due to

ultra-sensitivity of the assay and sample diluent optimization

IFN-α assay development

IF N -a

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

Av

era

ge

en

zy

me

s p

er b

ea

d

(A

EB

)

0 .1 1 1 0 1 0 0 1 0 0 0

0 .0 0 0 1

0 .0 0 1

0 .0 1

0 .1

1

1 0

L L O D : 0 .7 p g /m l

IFN-a

(Simoa)

IFN-a

(ELISA)

T r a n s fo rm o f IF N -a s td c u r v e

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

O.D

(4

50

nm

)

1 0 1 0 0 1 0 0 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

L L O D : 1 2 .5 p g /m l

LLOD: 12.5 pg/ml,

Range: 12.5-400 pg/ml

LLOD: 2 pg/ml,

Range: 2-500 pg/ml

6-fold improvement in

sensitivity

1. Capture bead

concentrate

• Buffer exchange the capture antibody, adjust its concentration

• Activate the beads with EDC

• Conjugate the antibody to the activated beads

• Determine the bead coating efficiency and bead aggregation profile

2. Detector antibodies

• Buffer exchange the detection antibody, adjust its concentration

• Conjugate with biotin

• Buffer-exchange the biotinylated antibody

• Determine the final detector concentration and reaction yield

3. Initial simoa testing

• Test if the capture and detection antibodies meet the criteria of the assay

• % monomeric beads > 75%

• Determine the concentration of the capture antibody that gives low background and high signal-to-noise ratio (0.3 / 0.5/ 0.7 mg/ml)

4. Optimize reagents

• Titrate the detector antibody concentration (0.1-1.2 ug/ml starting from 0.5 ug/ml)

• Titrate the SβG concentration (50-300 pM starting from 150 pM)

• Optimize the concentration of detector and SβG to reduce the background, increase the signal and get higher signal-to-noise ratio (Ideal background < 0.02, levels > 18 AEB should be avoided

• Perform spike-recovery and dilution experiments with the matrix and optimize the sample diluents accordingly

Chimeric Protein (X-Y)

C h im e r ic P ro te in (X -Y )

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

Av

era

ge

e

nz

ym

es

p

er b

ea

d

(A

EB

)

0 .1 1 1 0 1 0 0 1 0 0 0

0 .0 1

0 .1

1

1 0

1 0 0

C a p tu re : X , D e te c tio n : Y

C a p tu re : Y , D e te c tio n : X

Chimeric Protein (X-Y)