Ultra-Sensitive Cytokine and Biomarker

Assay Services

Stephen Parent, PhD

Director of Business Development

sparent@pblbio.com

(732) 777-9123 x117

IL-17

Ultrasensitive Cytokine Assay Services

using Singulex Erenna® Immunoassay System

Modified IA process

using magnetic beads

Single Molecule

Detection (SMD)

technology50- 1000 fold higher sensitivity over existing IA

products

More than 40 Assays Available

IL-17

Singulex Single Molecule Counting (SMC)

Erenna®

4.

5.

► Elution buffer

dissociates the

complex (analyte +

detection antibody

in simple buffer)

► Transfer to 384-

well plate

► Draw eluate into the

instrument for reading

► Fluorescently labeled

antibodies introduced

to laser confocal

microscope digital

counter

► Molecules are

individually detected

and digitized

Elution Molecule counting

Proprietary Singulex technology

Sample collection

2.

3.

► Transfer samples

to 96-well plate

► Incubate with

capture magnetic

antibody-coated

beads

► Wash to remove

unbound

antibodies

► Incubate with dye-

labeled detection

antibodies

1.

► Low abundance of

certain biomarkers in

complex biological

samples

Antibody incubation Washing & fluorescent labeling

Standard immunoassay protocol

More than 40 Assays Available

IL-17

Singulex Erenna® Assay Performance

Characterization

Parameters Evaluated

• Serum quantitation

• Plasma quantitation (other matrices upon request)• Assessment of anticoagulant effect

• Inter-assay precision (serum and plasma)

• Specificity of quantitation• Analyte specificity

• Serum and Plasma

• Assessed using alternate microparticles and polyclonal antibodies

• Linearity of Dilution• High analyte concentration

• Spiked analyte concentration

• Spike Recovery

IL-17

Singulex Erenna® Assay Performance

Characterization

Completed• IL-1b

• IL-6 (version 1)

• IL-17A (version 1)

• IL-17F (version 1)

• IL-17 A (version 2; plasma characterization in progress)

• IL-17F (version 2; plasma characterization in progress)

• IL-17 A/F Heterodimer

• IFNg

In Progress• TNFa

• IL-6 (version 2)

• cTnI

• GLP-1

Singulex Erenna® TNFa Assay Performance Characterization

(In Progress)

Parameter PBL Singulex

Reported

LLOQ (pg/ml) 0.10

0.20

Avg = 0.15

0.10

LOD (pg/ml) 0.002

0.053

Avg = 0.03

0.01

Y = 1517x + 102 R2 = 0.992

Sample Expected

(pg/ml)

Observed

(pg/ml)

% recovery

Serum 1 2.59 2.58 99.6%

Serum 2 2.63 2.45 93.2%

Quantification of TNFa in

Donor sera (median = 1.13 pg/ml)

and plasma (median = 0.35 pg/ml)

Spike recovery

Specificity:

Specificity was determined by changing

beads with concurrent alternate assay

Singulex Erenna® cTnI (v2) Assay Performance Characterization

(In Progress)

Parameter PBL Singulex

Reported

LLOQ (pg/ml) 0.35 0.40

LOD (pg/ml) 0.09 0.10

Y = 360x + 295 R2 = 0.971

Control Expected

(pg/ml)

Observed

(pg/ml)

% recovery

1 5.83 6.76 116%

2 47.57 49.39 104%

Quantification of sera

In cTnI assay

Performance of Assay Controls

Ultra-Sensitive Cytokine Assay

Services

Singulex Erenna IL-1b

Assay Performance Characterization

Specification IL-1b Goal Result

Quantitation of 20 NHS n = 23 Determine median

IL-1b concentration

Median [IL-1b] = 0.62 pg/ml

(not reported by Singulex)

Quantitation of 15 Plasma

(EDTA, citrate, heparin anti-

coagulant)

Determine median IL-1b

concentration and effect of

anti-coagulant

Median [IL-1b] = 0.06 – 0.20 pg/ml

(0.08 pg/ml reported by Singulex)

Inter-assay Precision <20% CV Serum: 3.1 – 22.9 % CV (mean = 13.2 ; n = 6)

Plasma: 0.0 – 55.7 % CV (mean = 16.7 ; n = 4)

Specificity:

bead swap

No cross-reactivity No cross-reactivity observed with antigen. Some variability with serum and plasma samples.

Specificity:

PAb knock-down

Reduction of assay signal Percent recovery 3.1% with antigen standard.

Percent recovery 6.9 – 8.6% with 3 of 4 serum

samples. Single sample near LLOQ 53%.

PAb not effective on plasma

Linearity of dilution 80-100% recovery 98.5 to 111.2 % for serum dilution

107.5% for plasma dilution

Spike Recovery 80-100% recovery 93.1% for spike recovery in serum

90.8% for spike recovery in plasma

Singulex Erenna® IL-1b Assay Performance

Characterization Summary

Singulex Erenna® IL-1b Assay Performance Characterization

Standard Curves and Observed Limits of Detection/Quantitation

Parameter PBL Singulex Reported

LLOQ

(pg/ml)

0.20

0.10

0.20

Avg = 0.17

0.10

LOD

(pg/ml)

0.040

0.018

0.050

Avg = 0.04

0.03

Y = 892x + 143 R2 = 0.997

Singulex Erenna® IL-1b Assay Performance Characterization

Median Donor Serum and Plasma Quantitation

Median Serum [IL-1b] = 0.62 pg/ml (n = 23 healthy)Median Heparin-Plasma [IL-1b] = 0.20 pg/ml

Median Citrate-Plasma [IL-1b] = 0.08pg/ml

Median EDTA-Plasma [IL-1b] = 0.06 pg/ml

Singulex = 0.08 pg/ml

Singulex Erenna® IL-1b Assay Performance Characterization

Specificity

Specificity:

Specificity was determined by changing beads with

concurrent alternate assay or incubating samples of

known concentration with IL-1b polyclonal antibody (not

optimized).

PAb does not appear to be effective in plasma samples.

Values close to the LLOQ (~ 0.2 pg/ml) may not

accurately reflect specificity in serum.

Sample ID [IL-1b] pg/ml % Recovery

Analyte Specificity

5 pg/ml Measured 6.06 ---

5 pg/ml + IL-17 A/F Beads 0.02 0.3%

5 pg/ml + IL-1b PAb 0.19 3.1%

Singulex Erenna® IL-1b Assay Performance Characterization

Specificity

Sample ID [IL-1b] pg/ml % Recovery

Serum Specificity

Endogenous 1

Endogenous 2

Endogenous 3

Endogenous 4

10.8

0.43

46.16

6.27

---

IL-1b PAb 1

IL-1b PAb 2

IL-1b PAb 3

IL-1b PAb 4

0.75

0.23

3.99

0.44

6.9%

53.5%

8.6%

7.0%

IL-17 A/F Beads 1

IL-17 A/F Beads 2

0.03

0.06

0.3%

14.0%

Plasma Specificity

Endogenous 1

Endogenous 2

Endogenous 3

0.46

0.23

2.76

---

IL-1b PAb 1

IL-1b PAb 2

IL-1b PAb 3

0.33

Not Detected

2.65

71.7%

0 %

97.0%

IL-17 A/F Beads 1

IL-17 A/F Beads 2

0.28

0.02

60.9%

8.7%

Specificity:

Specificity was determined by changing beads with concurrent alternate assay or incubating samples of known concentration with

IL-1b polyclonal antibody (not optimized). PAb does not appear to be effective in plasma samples.

Values close to the LLOQ (~ 0.2 pg/ml) may not accurately reflect specificity in serum.

Singulex Erenna® IL-1b Assay Performance Characterization

Inter-Assay Precision

Sample ID [IL-1b]

pg/ml

Day 1

[IL-1b]

pg/ml

Day 2

[IL-1b]

pg/ml

Day 3

% CV

574778S 0.15 0.16 4.6

574779S 39.9 31.0 46.16 19.6

574780S 4.88 6.27 17.6

574781S 0.34 0.29 11.2

574791S 0.31 0.43 22.9

574795S 11.3 10.8 3.1

651700P 2.58 2.67 4.8

651710P 0.21 0.23 6.4

651712P 0.20 0.46 55.7

651705P 0.10 0.10 0.0

Mean 13.2S

16.7P

Inter-Assay Precision:

IL-1b concentration quantified on separate days

S = Serum; P = Plasma

Singulex Erenna® IL-1b Assay Performance Characterization

Spike Recovery in Normal Healthy Donor Serum and Plasma

Serum 574779

Dilution

Observed Expected % Recovery

Neat 46.16

1:2 27.88 23.08 120.8

1:4 13.12 11.54 113.7

1:8 5.71 5.77 99.0

Mean (excluding neat, goal 80-120%) 111.2

Serum 574780

Dilution

Observed Expected % Recovery

Neat 6.27

1:2 2.92 3.14 93.0

1:4 1.61 1.57 102.5

1:8 0.78 0.78 100.0

Mean (excluding neat, goal 80-120%) 98.5

Plasma 651700

Dilution

Observed Expected % Recovery

Neat 2.76

1:2 1.32 1.38 95.6

1:4 0.87 0.69 126.1

1:8 0.34 0.34 100.0

Mean (excluding neat, goal 80-120%) 107.2

Sample Dilution:

Linearity of Dilution was performed by diluting serum

or plasma with a known concentration of IL-1b in

Standard Diluent.

IL-17

Ultrasensitive Biomarker Assay Services using

Quanterix Simoa™ Immunoassay System

Modified IA process

using magnetic beads

Single Molecule Array

technology

> 30 single analyte and multiplex assays available!

IL-17

Quanterix Simoa™ Immunoassay Technology