Anteo Diagnostics

Mix&GoTM

A General Method for Forming Functional

Protein Mono-Layers

August 2013

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

What Is Mix&Go?

A family of aqueous metal polymers bind to virtually all

surfaces used in life sciences

M

M

M M

M

Configurations

• 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

• 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.:

Forming Mix&Go Activated Surfaces

• 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

Microtiter plates:

Pipette 100 L

Mix&Go into

wells, incubate

for 30 minutes

and wash

Mix&Go

Add

nanoparticles

Mix well

Beads:

Activated in 30

mins and stable

for years

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

200 μg/mL 50 μg/mL 10 μg/mL

0

5,000

10,000

15,000

200 μg/mL 50 μg/mL 10 μg/mL 0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

200 μg/mL 50 μg/mL 10 μg/mL

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.

Increased Protein Stability

Stability studies using binding capacity of biotinylated mouse

Ab shows no difference even after 2 weeks at 37 C.

Freshly Coated 14 Days @ 25 C 14 Days @ 37 C

Streptavidin on Mix&Go Microarrays

Testing existing commercial products. Biotin-RPE binding on Streptavidin

Mix&Go microarray slides vs commercial standard (3D Type).

12.5 25 40 100 200

g/ml biotin-RPE 12.5 25 40 100 200

g/ml biotin-RPE

Top: Biotin-RPE binding on

Streptavidin Mix&Go slides

(far left) vs commercial

product (left).

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

12.5 25 40 100 200

g/ml biotin-RPE

Streptavidin on Mix&Go on Plates

0.000

0.500

1.000

1.500

2.000

2.500

0 100 200 300 400 500 600

OD

(450

-620n

m)

biotin Mouse Concentration (ng/mL)

Mix&Go on Greiner Low Bind Block

Mix&Go on Nunc Polysorp Block

Mix&Go on Corning Med Bind Stripwell

Thermo High Sensitivity Stripwell

Thermo Standard Block

Streptavidin Mix&Go plates (different brands) compared to Thermo/Pierce

Streptavidin plates. Streptavidin coating at 2 g/ml, 30 mins.

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.

Decreased Protein Usage (on Plates)

GM-CSF cAb titration on Mix&Go activated Greiner low binding plates

vs. passive binding on Nunc Maxisorp plates

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10

OD

(450

-620n

m)

cAb (ug/mL)

Mix&Go Greiner

Passive Nunc Maxisorp

Mix&Go Passive

cAb Conc 0.125 g/mL 1.0 g/mL

cAb Cost

per Plate* $1.10 $8.80

Plate Type Greiner

Low Bind

Nunc

Maxisorp

Plate Cost* $0.55 $4.92

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

Improving Antibody Distribution

Intra-plate CVs on TNFα sandwich assay. Comparison of

Mix&Go activation vs passive on Greiner Low Binding plates

A

D

G

0.8 1 1.2 1.4

1 2 3 4 5 6 7 8 9 10 11 12

Greiner Low Bind Plate - Mix&Go

1.2-1.4

1-1.2

0.8-1

Mix&Go

A

D

G

0.2

0.4

0.6

1 2 3 4 5 6 7 8 9 10 11 12

Greiner Low Bind Plate Passive

0.4-0.6

0.2-0.4

Passive

Mix&Go activation decreases well-to-well variation

while increasing activity compared to passive binding

on Greiner low binding plates

CV = 4.43% CV = 9.21%

Stability on Plates

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

1.800

Mix&Go on Greiner Medium

Stripwell

Passive on Greiner High Bind Stripwell

OD

(450

-620n

m)

IL-6 Stability Testing

Day 0

Day 8 37C

Day 15 37C

Temp = 37 C Mix&Go Passive

Day 0 vs.

Day 8 1.17% 8.64%

Day 0 vs.

Day 15 7.55% 17.75%

Change in activity from Day 0

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.

Advantages w/ Decreasing Surface

0.000

0.500

1.000

1.500

2.000

2.500

3.000

0 1,500 3,000 4,500 6,000 7,500 9,000 10,500

OD

(450

-620n

m)

Ag Concentration (pg/mL)

0.000

0.500

1.000

1.500

2.000

2.500

3.000

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.

Comparison of 96 well (Left) and 384 well (Right) micro-titre plates. TNFα

sandwich assay using cAb conc of 0.5 g/ml

Mix&Go

No Mix&Go

Coating on Other COC/COP Plastics

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.

0

0.5

1

1.5

2

2.5

3

COC M&GC10

O.D

. (4

50 n

m)

24 hours

TNF-a

TnI

TSH

0 20 40 60 80 1000.0

0.5

1.0

Time (min)

O.D

.

TNFa

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.

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.

5

4

3

2

1

0µ

m

543210

µm

-6

-4

-2

0

2

4

6

nm

5

4

3

2

1

0

µm

543210

µm

-10

-5

0

5

10

nm

Gold nanoparticles on Mix&Go

activated COC Gold nanoparticles on COC

by passive binding

Surface coverage of gold colloids on COC by passive binding

was calculated to be 11%. Mix&Go COC surfaces gave 64%.

Sandwich Assays on Magnetic Particles

IFN-ϒ chemiluminescense assay on Mix&Go MyOne (Life Technologies)

vs. MyOne Tosyl magnetic particles.

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

MyOne Mix&Go Dynabeads

MyOne Tosyl Dynabeads

RL

U (

Go

at

an

ti M

ou

se Ig

G -

HR

P)

Loading Assay: Mix&Go vs. Tosyl Beads

Antibody per mg beads

Mix&Go: 15ug Ab

Tosyl: 40ug Ab

-

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

180,000

0 500 1000 1500 2000 2500 R

LU

IFN-ϒ antigen pg/mL

Sensitivity Assay: Mix&Go vs. Tosyl Beads

15ugAb+15ugBSA/mg MyOne Mix&Go beads

40ugAb/mg MyOne Tosyl beads

Signal to Noise (S/N)

Mix&Go: 2607

Tosyl: 1787

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.

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 M-

270 Dynabeads

Right two tubes: Antibody M-280

Tosyl Dynabeads

Mix&Go beads form cleaner bead plugs under a magnetic field than

Tosyl beads.

Bead Handling Characteristics

*Tosyl beads coupled at recommended concentrations

Streptavidin on 200 nm Latex Nanoparticles

Comparison of Streptavidin on 243 nm Mix&Go activated particles

with Thermo’s Power-Bind Streptavidin 294 nm particles

Streptavidin on Mix&Go activated latex nanoparticles shows over 5x increase

in biotinylated antibody loading factoring in particle size.

Non Specific Binding

Goat anti Mouse IgG–HRP

-

2,000,000

4,000,000

6,000,000

8,000,000

10,000,000

12,000,000

14,000,000

16,000,000

18,000,000

Streptavidin Mix&Go 243nm Particles

Commercial Streptavidin Thermo Power-Bind (294nm) Particles

RLU

(B

iotin

yla

ted

Mo

use

Ig

G/G

AM

-H

RP

)

Batch 1

Batch 2

0

5,000

10,000

15,000

20,000

25,000

30,000

Streptavidin Mix&Go 243nm Particles

Commercial Streptavidin Thermo Power-Bind (294nm) Particles

RLU

(G

oa

t a

nti M

ou

se

Ig

G -

HR

P)

Batch 1

Batch 2

Specific Binding

Biotinylated Mouse IgG/Goat anti Mouse

IgG–HRP

Colloidal stability of nanoparticles after Mix&Go activation and

protein coupling is maintained with particles below 40 nm.

Mix&Go activated Ademtech

beads coupled with 560 mAb

(125ug Ab/mg beads) in

pooled plasma

Antibody on Mix&Go coated magnetic nanoparticles (500 nm).

Brownian motion under magnetic field in pooled plasma (TnI

assay) http://youtu.be/UB7mctqmP-c

Maintaining Brownian Motion

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.

400 500 600 700 800

OD

No

rma

lize

d

Wavelength (nm)

Mix&Go

Passive

400 500 600 700 800

OD

No

rma

lize

d

Wavelength (nm)

Mix&Go + IgG

Passive + IgG

Mix&Go coating resulted in a LSPR

λ-max shift of 4nm with improved

colloid dispersity.

On Ab addition, there is a further λ-max shift

of 9nm on Mix&Go gold colloids. Without,

Mix&Go, the colloids aggregate.

Improving the Flow of Nanoparticles

TEM analysis of Ab bound to

Mix&Go iron oxide nanoparticles

(10-15 nm).

Mix&Go No Mix&Go

The hydrophilic nature of Mix&Go particles allow better “flow” in

aqueous environments giving faster pull down speeds under magnetic

field.

Abs Particle

Antibody on Mix&Go Magnetite

ESC-130416-1

Two step functionalisation of super

paramagnetic nanoparticles;

Mix&Go addition, wash than

antibody addition.

Chemiluminescent loading assay.

Detection with GAM-HRP (@

0.005ug/ml). Lumigen PS-atto was

used as the substrate.

The very large surface area of

magnetite nanoparticles give very

large protein binding capacity

giving high Signal to Background

of 3,106:1

41,501,039

13,360

-

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

30,000,000

35,000,000

40,000,000

45,000,000

Specific Signal Background

RL

U (

GA

M-H

RP

)

Assay Readout

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.

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

Contact Information