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Anteo Diagnostics
Mix&Go

TM

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 ve...
What Is Mix&Go?
A family of aqueous metal polymers  bind to virtually all
surfaces used in life sciences
Configurations
M...
Forming Mix&Go Activated Surfaces
•

•

Mix&Go is a cationic polymer: mechanism of binding is via both
electrostatic and c...
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 Microarrays
Testing existing commercial products. Biotin-RPE binding on Streptavidin
Mix&Go microar...
Streptavidin on Mix&Go on Plates
Streptavidin Mix&Go plates (different brands) compared to Thermo/Pierce
Streptavidin plat...
Decreased Protein Usage (on Plates)
GM-CSF cAb titration on Mix&Go activated Greiner low binding plates
vs. passive bindin...
Improving Antibody Distribution
Intra-plate CVs on TNFα sandwich assay. Comparison of
Mix&Go activation vs passive on Grei...
Stability on Plates
Change in activity from Day 0
IL-6 Stability Testing
1.800

Temp = 37 C

Mix&Go

Passive

Day 0 vs.
Da...
Advantages w/ Decreasing Surface
Comparison of 96 well (Left) and 384 well (Right) micro-titre plates. TNFα
sandwich assay...
Coating on Other COC/COP Plastics
Loading assay using capAb (1 g/ml) and detection using GAM-HRP
(0.1 g/ml) on passively b...
Gold Colloids on Mix&Go COC Surfaces
Mix&Go can bind more than just proteins. AFM images of gold colloids
(40 nm) on COC a...
Sandwich Assays on Magnetic Particles
IFN-ϒ chemiluminescense assay on Mix&Go MyOne (Life Technologies)
vs. MyOne Tosyl ma...
Bead Handling Characteristics
Mix&Go MyOne vs. Tosylated MyOne*

Left two tubes: Antibody Mix&Go
MyOne Dynabeads
Right two...
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

Bat...
Maintaining Brownian Motion
Antibody on Mix&Go coated magnetic nanoparticles (500 nm).
Brownian motion under magnetic fiel...
Mix&Go on Gold Colloids
Localized Surface Plasmon Resonance (LSPR) studies on gold colloids
(Sigma, 100 nm). Mix&Go forms ...
Improving the Flow of Nanoparticles

Mix&Go

No Mix&Go

Particle

Abs

TEM analysis of Ab bound to
Mix&Go iron oxide nanop...
Antibody on Mix&Go Magnetite
45,000,000

Two step functionalisation of super
paramagnetic nanoparticles;
Mix&Go addition, ...
Benefits of Mix&Go
• Forms stable activated surface on planer surfaces of glass, ceramics
or engineering plastics as well ...
Contact Information
Thank you very much for your interest in Mix&Go.
For more information please contact Anteo directly:

...
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Mix&Go Surface Chemistry - How to Easily Attach a Protein to a Synthetic Surface

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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

Published in: Health & Medicine, Technology
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Mix&Go Surface Chemistry - How to Easily Attach a Protein to a Synthetic Surface

  1. 1. Anteo Diagnostics Mix&Go TM A General Method for Forming Functional Protein Mono-Layers August 2013
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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

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