Ultrasensitive cytokine and biomarker assay services provided by PBL Assay Science

1. Ultra-Sensitive Cytokine and Biomarker
Assay Services
Stephen Parent, PhD
Director of Business Development
sparent@pblbio.com
(732) 777-9123 x117

2. IL-17
Ultrasensitive Cytokine Assay Services
using Singulex Erenna® Immunoassay System
Modified IA process
using magnetic beads
Single Molecule
Detection (SMD)
technology
50- 1000 fold higher sensitivity over existing IA
products
More than 40 Assays Available

3. 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

4. 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

5. 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

6. 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

7. 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

8. Ultra-Sensitive Cytokine Assay
Services
Singulex Erenna IL-1b
Assay Performance Characterization

9. 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

10. 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

11. 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

12. 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%

13. 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.

14. 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

15. 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.

16. 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!

17. IL-17
Quanterix Simoa™ Immunoassay Technology