Metal Polymers, A Glue to Immobilise Proteins Onto Synthetic Surfaces
Abernethy N, Chung E, Fontanelle BT, Gao Y, Jennins D, Koudijs MM, Lim D, Yang L, Ling T, Vukovic P, Wong A, Maeji, NJ. Anteo Diagnostics Ltd, Brisbane, Australia; www.anteodx.com
Introduction
Results
Results, Cont.
Many different applications such as bio-separations, biomaterials, drug delivery, biomarker discovery
involve the immobilisation of biological materials onto synthetic surfaces. The challenge is that most
biological materials such as proteins are easily damaged when attached to some synthetic surface. This
challenge is further compounded by the variety of synthetic surfaces/materials as well as the variety of
biological materials that need to be localised on such surfaces.
Key Characteristics of Metal Polymers
Resulting Benefits of Metal Polymers in Immunoassays
We studied the characteristics of metal polymers, such as Mix&Go, when used to attach proteins, e.g.
antibodies, streptavidin, and Proteins A or G to synthetic surfaces with focus on characteristic that would
improve immunoassay performance or ease-of use:
Sensitivity and Dynamic Range: Under limited surface area for immobilising capture antibodies, e.g.
FACS, significant sensitivities can be obtained
The objective of our work was to develop a surface chemistry which maintains protein function and
orientation per unit surface area, regardless of the surface used and in addition
Fig.3. Binding GAM
antibodies to
microarray slides
1.0
15000
1.000
10000
0.900
0.800
5000
0.700
Si-OH
NSB
The approach we took was to use metal chelation with avidity binding concepts, i.e., create multiple,
weak binding forces that together form strong but gentle binding interactions.
GAM
ProMag
NSB
GAM
OD 450
0.600
0
Methods
0.1
100,000
50,000
0.400
0.300
0
M&G
passive
0.100
Mix&Go
0.000
0
5
10
15
20
Water
LoProdyne
25
Mix&Go
Water
UltraBind
Time (min)
Fig 6. Binding GAM antibodies to
hydroxy-methylmethacrylate
membranes
Fig. 5. Comparison of Mix&Go vs.
passive adsorption on PS Greiner,
binding TSH capAb at 500 ng/ml (50
ng/well).
Fig. 3 Mix&Go on a variety of surfaces
In Immobilized Metal Affinity Chromatography (IMAC), metal ions such as Ni(III) and Co(III) are used to
isolate and purify His tagged proteins. In order to do so, the metal ion needs to bind to particular ligands
(such as NTA) that are coupled to the synthetic surface. Even so, protein binding strength is relatively
weak even for His tag proteins ( M binding strength).
Using high throughput screening methods, we identified various metal ion, counter ion combinations that
exhibited far stronger binding potential to proteins (Anal. Biochem. 363 (2007) 97-107) even without the
need for special ligands such as NTA (on the synthetic surface) or His tags (on the protein). One such
combination is the use of metal ions in polymeric form. Metal ions can be linked together with the use of
various multi-valent chelating ligands and some metal ions can naturally form hydrolytic polymers.
Mix&Go(TM) is one such family of polymeric metal ions based around Cr(III).
Metal polymers such as Mix&Go are water soluble and because it can exist as di-mers, tri-mers and
other longer polymers, its character gives Mix&Go multiple points of chelation whose number increases
overall binding strength. In addition, polymer structure may potentially impart some degree of selectivity
according to its shape.
Stable Binding of Proteins: MixGo has been proven to be stable in conditions such as:
 Boiling beads for 10 mins in 2% SDS-PAGE sample buffer (SDS, DTT, Glycerol,Coomassie Blue
dye)
 Freeze-Drying Mix&Go-activated Beads for over a month
 Treating beads for 48 hours with 0.5M EDTA then boiling them in SDS-PAGE buffer
 Storing Mix&Go-activated beads in 200mM EDTA for 1 year
 Extremes of pH ~ 2.5 (Glycine or Citrate buffer elution) to pH ~ 12 (0.5M NaOH washing)
M
M
M
M
M M
M
M M
M
M
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
Mix&Go
0.2
24
0.0006
3.5
0.5
12
Tosyl
2.4
1040
0.0017
200
0.9
67
Improvement
Factor
60
43
2.8
57
1.8
5.6
Purification of human IgG with Protein A beads
Dynabead Control
Mix&Go
8.5
12.2
Binding Capacity for IgG, related to measured bead
concentration (mg/gm bead)
Yield of IgG in elution step
67%
Goat IgG/Mix&Go
Sheep IgG/Amide
0
5
10
15
20
ug of Ab/mg beads
25
Fig. 7. Binding of biotinylated
rat IL6 Ab to Streptavidin
Mix&Go plates (Greiner and
Nunc PolySorp) using anti-rat
Fab specific detection is far
more efficient than with
standard commercially
available Streptavidin plates.
Mix&Go plates achieved
detection of <2 ng/ml
1.200
1.000
0.800
0.600
Streptavidin Mix&Go plate 1 - Goat anti Rat IgG F(ab')2
Streptavidin Mix&Go plate 2 - Goat anti Rat IgG F(ab')2
Commercial Streptavidin plate - Goat anti Rat IgG F(ab')2
0.000
0
20
40
60
Biotinylated - IL-6 mAb ng/mL
80
100
120
35
1
Fig.9. IL6 Ab (as detected by
anti-rat Fab specific Ab) on
Mix&Go surfaces compared to
passive adsorption on microtitre
plates.
0.9
0.8
0.7
0.6
0.5
0.4
M&G
0.3
passive
0.1
0
0
200
400
600
800
1000
1200
Rat anti- Human IL-6 cAb (ng/ml)
Linear Correlation: Under non-saturation conditions the amount of antibody added equals the amount
of active antibody on the surface.
6000
y = 20.05x + 6.2406
R² = 0.9666
TnI B1 mAb
TnI B2 mAb
y = 19.993x - 80.251
R² = 0.9822
TSH mAb
4000
y = 24.786x - 67.892
R² = 0.9705
3000
2000
y = 21.064x - 171.34
R² = 0.99
Mouse IgG pAb
Fig.10. With Mix&Go, all 4
antibodies show a high linear
correlation between what is added
and what is immobilised on MyOne
Dynal beads.
Linear (TnI B1 mAb)
Linear (TnI B2 mAb)
Linear (TSH mAb)
1000
1.400
OD 450
30
Time to Result: Increased density of functional antibodies improves speed of binding cognate ligands,
especially under limiting situations we observer far more functional, correctly orientated bound antibody
92%
1.600
0.200
GM-CSF
Goat IgG/Amide
5000
0.400
TNF
Fig.8 Loading efficiency for 2
different IgGs comparing amide
vs. Mix&Go coupling on Dynal
M270 beads.
Sheep IgG/Mix&Go
Good functionality of bound Proteins: Despite fast and strong binding, proteins bound to Mix&Go
surfaces are gentle that nearly all immobilise proteins are functional.
Fig 2. Different metal polymer shapes are
possible and depending on metal ion
number and shape, can give a large range of
binding strengths and potentially selectivity
to some target protein.
Since all sorts of basic, neutral and acidic groups can chelate with metal ions, these metal polymers
were investigated as a generic “glue” to bind proteins to all sorts of synthetic surfaces. For ease of use,
synthetic surfaces were first coated with these metal polymers to form an activated but stable surfaces.
These Mix&Go activated surfaces were assessed for their potential to bind all sorts of proteins to many
different surfaces.
PSA
0.2
Table 1. In immunoprecipitation studies >90% of immobilised Protein A bound and subsequently eluted IgG.
M
TSH
Nil
150,000
0.500
0.200
Fig.4. Binding GAM
antibodies to silica and
polymer beads
HBsAg
200,000
OD 450
20000
IL-6*
Reducing Antibody Use: Because of rapid quantitative coupling there is less antibody wastage. In
bead-based immunoassays we demonstrated up to 90% antibody savings, and 67% savings of beads at
comparable sensitivity.
IgG binding (RLU)
Improve sensitivity and dynamic range
Reduce antibody usage
Increase speed of assays
Provide consistent performance even with “problem antibodies” that are sensitive to passive
binding or existing covalent chemistry.
Loading of Mouse IgG-fluorescein (MFI)




Activation
Table 2 Loading efficiency for 2 different IgGs comparing amide vs. Mix&Go
coupling on industry standard Tosyl Beads
MFI (GAM-FITC)
Realistic Situation
Goal
Flexibility in Surfaces: Mix&Go will bind strongly to any surface having electron donating potential to
form a stable activated surface for protein binding. Surfaces tested include:
 Acid, amine and non-functionalised polymer beads
 Bare silica beads or slides
 Sepharose, polyvinylalcohol, metal oxides, etc.
 Plain polystyrene plates
 Plasma or gamma treated materials that do not normally bind any proteins
MFI (Mouse IgG - FITC)
Fig.1. Immunoassays are one of the major
techniques requiring attachment of proteins, e.g.
antibodies to synthetic surfaces. In standard
ELISA tests the damage done to a portion of the
antibodies might not limit sensitivity because
antibodies are present in vast excess. This,
however, is no longer the case with increasing
miniaturisation where the available surface area
to bind proteins becomes limiting and every
antibody counts.
Immunoassay Analyte
Reaction Kinetics: Binding Mix&Go to synthetic surfaces and binding proteins to Mix&Go activated
surfaces are both very fast with maximum loading essentially achieved within 1 min.
Linear (Mouse IgG pAb)
0
0
50
100
150
200
250
ug of antibody/mL
Conclusion
Mix&Go is a family of metal polymers of different sizes and shapes. The key benefits of using such
metal polymers to attach proteins to synthetic surfaces are simplicity, as well as faster and simpler
assays with greater sensitivity and improved dynamic range. Especially for miniaturized formats where
maintaining correct antibody orientation, good antibody distribution, and activity of each antibody are
critical, these metal polymer “glues” will provide enhanced performance and ease-of-use.