overview document showing how scientist can easily and quickly use Anteo's Mix&Go surface coating to attach biomolecules, e.g. antibodies, streptavidin, Protein A to a variety of synthetic surfaces from nanobeads to glass slides withut causing damage to the biomolecule

1. Anteo Diagnostics
Mix&Go
TM
A General Method for Forming Functional
Protein Mono-Layers
August 2013

2. Developing a Universal “Glue”
• What is Mix&Go?
• aqueous metal polymers which use multiple weak binding
forces to form very strong bonds.
• Mix&Go surfaces are;
• stable for years but in the presence of proteins rapidly bind
with minimal conformational damage.
• acts as a molecular “velcro” for proteins, synthetic polymers,
nanoparticles, etc.
• Characteristics of proteins bound to Mix&Go surfaces are:
• functionally active protein mono-layers
•
Resulting benefits of Mix&Go are:
• many and varied

3. What Is Mix&Go?
A family of aqueous metal polymers  bind to virtually all
surfaces used in life sciences
Configurations
M
M
M
M
M
• Metal oligomers with different
shape and charge densities
• Determines speed and
strength of binding to surfaces
and biomolecules, e.g. proteins
Mix&Go is based on Cr(III)
• Slow exchange half life; order of hours.
• Not considered a health risk from numerous studies, e.g.,
International Agency for Research on Cancer (IARC)
• Not considered hazardous waste by US EPA
• An essential elements in human health

4. Forming Mix&Go Activated Surfaces
•
•
Mix&Go is a cationic polymer: mechanism of binding is via both
electrostatic and co-ordination forces
Making Mix&Go surfaces is easy, e.g.:
Microtiter plates:
Pipette 100 L
Mix&Go into
wells, incubate
for 30 minutes
and wash
•
Beads:
Mix&Go
Add
nanoparticles
Mix well
Activated in 30
mins and stable
for years
Mix&Go is reactive with:
• Acid, amine, hydroxy, epoxy and other polymer beads
• Nanoparticles: gold colloids, iron oxides, etc.
• Sepharose, poly(vinylalcohol), poly(hydroxyethylmethacrylate)
• Glass, silica oxides, titanium oxides, ceramics, etc.
• Polystyrene microtitre plates
• Engineering thermoplastics such as COC/COP

5. Increased Protein Stability
Streptavidin microarray slides need to be stored at -20°C and have shelf
life of hours at RT. Streptavidin on Mix&Go coated microarray slides
show excellent stability even after 14 days at 25 C and 37 C.
18,000
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
Freshly Coated
200 μg/mL
50 μg/mL
10 μg/mL
18,000
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
14 Days @ 25 C
15,000
14 Days @ 37 C
10,000
5,000
0
200 μg/mL
50 μg/mL
10 μg/mL
200 μg/mL
Stability studies using binding capacity of biotinylated mouse
Ab shows no difference even after 2 weeks at 37 C.
50 μg/mL
10 μg/mL

6. Streptavidin on Mix&Go Microarrays
Testing existing commercial products. Biotin-RPE binding on Streptavidin
Mix&Go microarray slides vs commercial standard (3D Type).
Top: Biotin-RPE binding on
Streptavidin Mix&Go slides
(far left) vs commercial
product (left).
12.5 25 40 100 200
12.5 25 40 100 200
g/ml biotin-RPE
g/ml biotin-RPE
12.5 25 40 100 200
g/ml biotin-RPE
Bottom: Same experiment
with prior biotin blocking
shows no binding on Mix&Go
slides (far left). Commercial
product binding is all Non
Specific Binding (NSB)
(left).
12.5 25 40 100 200
g/ml biotin-RPE

7. Streptavidin on Mix&Go on Plates
Streptavidin Mix&Go plates (different brands) compared to Thermo/Pierce
Streptavidin plates. Streptavidin coating at 2 g/ml, 30 mins.
2.500
Mix&Go on
Greiner Low
Bind Block
2.000
OD (450-620nm)
Mix&Go on
Nunc Polysorp
Block
1.500
Mix&Go on
Corning Med
Bind Stripwell
1.000
Thermo High
Sensitivity
Stripwell
0.500
Thermo
Standard Block
0.000
0
100
200
300
400
biotin Mouse Concentration (ng/mL)
500
Using only 200 ng/well and 30 min coating time,
streptavidin coated onto Mix&Go plates perform as well or
better than existing commercial products.
600

8. Decreased Protein Usage (on Plates)
GM-CSF cAb titration on Mix&Go activated Greiner low binding plates
vs. passive binding on Nunc Maxisorp plates
2.5
Mix&Go
cAb Conc
OD (450-620nm)
1.0 g/mL
$1.10
$8.80
Plate Type
Greiner
Low Bind
Nunc
Maxisorp
Plate Cost*
1.5
0.125 g/mL
cAb Cost
per Plate*
2
Passive
$0.55
$4.92
1
Mix&Go
Greiner
Passive Nunc
Maxisorp
0.5
0
0
2
4
6
cAb (ug/mL)
8
10
The binding kinetics of Mix&Go activated polystyrene surface
results in peak signal at lower antibody concentration and plateaus
regardless of increased concentration.
Significant COGS savings can be achieved with Mix&Go

9. Improving Antibody Distribution
Intra-plate CVs on TNFα sandwich assay. Comparison of
Mix&Go activation vs passive on Greiner Low Binding plates
Mix&Go
Passive
Greiner Low Bind Plate Passive
Greiner Low Bind Plate - Mix&Go
1.2-1.4
0.4-0.6
1-1.2
0.2-0.4
0.8-1
A
D
1.4
1.2
1
0.8
12
10 11
G
8 9
6 7
4 5
3
CV = 4.43%
1 2
A
0.6
0.4
0.2
D
11 12
9 10
G
7 8
5 6
3 4
1 2
Mix&Go activation decreases well-to-well variation
while increasing activity compared to passive binding
on Greiner low binding plates
CV = 9.21%

10. Stability on Plates
Change in activity from Day 0
IL-6 Stability Testing
1.800
Temp = 37 C
Mix&Go
Passive
Day 0 vs.
Day 8
1.17%
8.64%
Day 0 vs.
Day 15
7.55%
17.75%
1.600
1.400
Day 0
OD (450-620nm)
1.200
1.000
Day 8 37C
0.800
Day 15 37C
0.600
0.400
0.200
0.000
Mix&Go on
Greiner Medium
Stripwell
Passive on
Greiner High
Bind Stripwell
Accelerated stability @ 15 days
stored at 37 C equates to
>1year stability stored at 4 C
Mix&Go C10 activated plates
coated with IL-6 and blocked
with 10mM PBS + 1% BSA +
5% Sucrose
Mix&Go plates maintain protein stability better than passive binding.

11. Advantages w/ Decreasing Surface
Comparison of 96 well (Left) and 384 well (Right) micro-titre plates. TNFα
sandwich assay using cAb conc of 0.5 g/ml
3.000
3.000
2.500
2.500
2.000
2.000
1.500
1.500
1.000
1.000
OD (450-620nm)
Mix&Go
No Mix&Go
0.500
0.500
0.000
0.000
0
1,500 3,000 4,500 6,000 7,500 9,00010,500
Ag Concentration (pg/mL)
0
1,500 3,000 4,500 6,000 7,500 9,000 10,500
Ag Concentration (pg/mL)
Antibody conformation and orientation are preserved resulting in more
functional antibodies. The effect is more pronounced on smaller surface
areas where each antibody becomes more important.

12. Coating on Other COC/COP Plastics
Loading assay using capAb (1 g/ml) and detection using GAM-HRP
(0.1 g/ml) on passively bound vs. Mix&G activated COC surfaces.
24 hours
TNFa
1.0
3
O.D. (450 nm)
O.D.
2.5
0.5
0.0
2
TNF-a
1.5
TnI
TSH
1
0.5
0
20
40
60
Time (min)
80
100
0
COC
M&GC10
Capture antibody binding efficiency to Mix&Go activated COC
surfaces is far faster than passive binding. Even after 24 hr
coating, passive binding did not reach Mix&Go levels.

13. Gold Colloids on Mix&Go COC Surfaces
Mix&Go can bind more than just proteins. AFM images of gold colloids
(40 nm) on COC and Mix&Go activated COC surfaces.
Gold nanoparticles on Mix&Go
activated COC
Gold nanoparticles on COC
by passive binding
5
5
4
10
6
4
4
5
2
µm
0
nm
µm
3
2
0
2
-2
-5
1
-4
1
-10
-6
0
0
1
2
3
µm
4
5
0
0
1
2
3
4
5
µm
Surface coverage of gold colloids on COC by passive binding
was calculated to be 11%. Mix&Go COC surfaces gave 64%.
nm
3

14. Sandwich Assays on Magnetic Particles
IFN-ϒ chemiluminescense assay on Mix&Go MyOne (Life Technologies)
vs. MyOne Tosyl magnetic particles.
Loading Assay: Mix&Go vs. Tosyl Beads
160000
140000
180,000
Antibody per mg beads
Mix&Go: 15ug Ab
Tosyl: 40ug Ab
120000
15ugAb+15ugBSA/mg MyOne Mix&Go beads
160,000
40ugAb/mg MyOne Tosyl beads
140,000
120,000
RLU
RLU (Goat anti Mouse IgG - HRP)
180000
Sensitivity Assay: Mix&Go vs. Tosyl Beads
100000
100,000
80000
80,000
60000
60,000
40000
40,000
20000
20,000
Signal to Noise (S/N)
Mix&Go: 2607
Tosyl: 1787
-
0
MyOne Mix&Go
Dynabeads
MyOne Tosyl Dynabeads
0
500
1000
1500
IFN-ϒ antigen pg/mL
2000
S/N of Mix&Go MyOne beads is superior to MyOne Tosyl beads using
less than half the amount of capture antibody. No co-coupling reagent
such as BSA were used.
2500

15. Bead Handling Characteristics
Mix&Go MyOne vs. Tosylated MyOne*
Left two tubes: Antibody Mix&Go
MyOne Dynabeads
Right two tubes: Antibody MyOne
Tosyl Dynabeads
Left two tubes: Antibody Mix&Go M270 Dynabeads
Right two tubes: Antibody M-280
Tosyl Dynabeads
Mix&Go beads form cleaner bead plugs under a magnetic field than
Tosyl beads.
*Tosyl beads coupled at recommended concentrations

16. Streptavidin on 200 nm Latex Nanoparticles
18,000,000
30,000
16,000,000
14,000,000
12,000,000
Batch 1
10,000,000
Batch 2
8,000,000
6,000,000
4,000,000
2,000,000
RLU (Goat anti Mouse IgG - HRP)
RLU (Biotinylated Mouse IgG/GAMHRP)
Comparison of Streptavidin on 243 nm Mix&Go activated particles
with Thermo’s Power-Bind Streptavidin 294 nm particles
Batch 1
25,000
Batch 2
20,000
15,000
10,000
5,000
0
Streptavidin Mix&Go 243nm
Particles
Commercial Streptavidin Thermo
Power-Bind (294nm) Particles
Specific Binding
Biotinylated Mouse IgG/Goat anti Mouse
IgG–HRP
Streptavidin Mix&Go 243nm
Particles
Commercial Streptavidin Thermo
Power-Bind (294nm) Particles
Non Specific Binding
Goat anti Mouse IgG–HRP
Streptavidin on Mix&Go activated latex nanoparticles shows over 5x increase
in biotinylated antibody loading factoring in particle size.

17. Maintaining Brownian Motion
Antibody on Mix&Go coated magnetic nanoparticles (500 nm).
Brownian motion under magnetic field in pooled plasma (TnI
assay) http://youtu.be/UB7mctqmP-c
Mix&Go activated Ademtech
beads coupled with 560 mAb
(125ug Ab/mg beads) in
pooled plasma
Colloidal stability of nanoparticles after Mix&Go activation and
protein coupling is maintained with particles below 40 nm.

18. Mix&Go on Gold Colloids
Localized Surface Plasmon Resonance (LSPR) studies on gold colloids
(Sigma, 100 nm). Mix&Go forms a thin film (<1 nm) on which binds a
mono-layer of proteins.
OD Normalized
Mix&Go
400
500
600
700
Mix&Go +
IgG
Passive +
IgG
OD Normalized
Passive
800
Wavelength (nm)
Mix&Go coating resulted in a LSPR
λ-max shift of 4nm with improved
colloid dispersity.
400
500
600
Wavelength (nm)
700
800
On Ab addition, there is a further λ-max shift
of 9nm on Mix&Go gold colloids. Without,
Mix&Go, the colloids aggregate.

19. Improving the Flow of Nanoparticles
Mix&Go
No Mix&Go
Particle
Abs
TEM analysis of Ab bound to
Mix&Go iron oxide nanoparticles
(10-15 nm).
The hydrophilic nature of Mix&Go particles allow better “flow” in
aqueous environments giving faster pull down speeds under magnetic
field.

20. Antibody on Mix&Go Magnetite
45,000,000
Two step functionalisation of super
paramagnetic nanoparticles;
Mix&Go addition, wash than
antibody addition.
41,501,039
40,000,000
35,000,000
RLU (GAM-HRP)
30,000,000
Chemiluminescent loading assay.
Detection with GAM-HRP (@
0.005ug/ml). Lumigen PS-atto was
used as the substrate.
25,000,000
20,000,000
The very large surface area of
magnetite nanoparticles give very
large protein binding capacity
giving high Signal to Background
of 3,106:1
15,000,000
10,000,000
5,000,000
13,360
Specific Signal
Background
Assay Readout
ESC-130416-1

21. Benefits of Mix&Go
• Forms stable activated surface on planer surfaces of glass, ceramics
or engineering plastics as well as beads and nanoparticles.
Benefits:
• universal, extremely stable coating for almost all surfaces
• water based chemistry is easy to use and manufacture
• 1 nm coating does not interfere with detection
• Forms undamaged protein mono-layer.
Benefits:
• Less non-specific binding
• Increased performance/sensitivity, esp. with limited surface area
• No need for anti-species Abs or Streptavidin constructs to bind
antibodies onto surfaces.
• Less protein and/or beads required.
Benefits:
• Reduced COGS
• Saves valuable/rare antibodies
• Improved reproducibility bead-to-bead, well-to-well, batch-to-batch.
Benefit:
• Improved accuracy with improved %CV.

22. Contact Information
Thank you very much for your interest in Mix&Go.
For more information please contact Anteo directly:
Headquarters
Anteo Diagnostics Ltd
Suite 4, 26 Brandl Street
Brisbane Technology Park
Eight Mile Plains, QLD 4113
Australia
Geoff Cumming, CEO
Geoff.cumming@anteodx.com
P:+61-417-203-021
US Contact
Tina Baumgartner
Tina.baumgartner@anteodx.com
P: +1-510-508-8462