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Hassle Free Assay Development Through the Use of Avidity Binding Metal Chelation Surface Coating
Jennins D, Vukovic P, Gao Y, Huang C, Baumgartner T, Carroll M, Chung E, Cooper S, de las Heras R, Hodyl J, Ling T, Maeji J, McElnea C, Munian C, Ohse B, Wong A, Yang L
Anteo Technologies Pty Ltd | Eight Mile Plains, Brisbane, Australia | www.anteotech.com
Introduction
Attaching biomolecules onto solid supports is a necessary step in developing immunoassays for disease research, such as cancer or infectious diseases. However, direct immobilisation of antibodies to synthetic
surfaces can damage proteins and adversely impact both their structure and function. The conventional methods, for example passive adsorption and covalent binding, are continually challenged by new generation
materials, miniaturisation, and increasingly complex assay platforms. Anteo Technologies has developed an alternative approach, Mix&Go™, that utilises a metal polymer surface chemistry. The polymeric metal ions of
Mix&Go, chelate and bind by avidity to both the surface and to biomolecules, acting as a molecular velcro (Figure 1). The AMG Activation and Coupling Kits utilise Mix&Go technology to allow for rapid generation of
coupled particles. Mix&Go gives the user the ability to control the loading of antibody (Figure 2). It also allows the coupling of other binding reagents such as streptavidin, enzymes or even other particles – all at the
same time.
Figure 1. Mix&Go, a molecular glue comprised of polymeric metal
ions that chelate to available electron donating groups on synthetic
surfaces and proteins. Figure 2. The process of activation and coupling using Mix&Go
Aim – Immunoprecipitation Using the AMG Coupling Kit
The AMG Coupling Kit, 1 µm Magnetic Particles, was used to prepare particles that easily purified target
molecules from cell lysate. Commonly used Protein A and Protein G coupled particles will specifically
bind antibodies for target purification. A problem with this approach is that the antibody will elute along
with the target protein. The aim is to show, using the AMG Coupling Kit, that the antibody does not
simultaneously elute with the target.
Method – Coupling Kit
The AMG Coupling Kit, 1 µm Magnetic Particles comes with pre-activated 1 µm particles and the buffers
required to couple proteins of choice. Anti-ERK (Merck Millipore) monoclonal antibody was coupled to
the particles in the kit using the provided instructions. The particles in the coupling kit are already
activated, making it possible for the user to simply couple their protein.
Results – Coupling Kit
Antibody (mono- or polyclonal) directly coupled to Mix&Go particles have been used for
immunoprecipitation (IP) and purification. Figure 3 and 4 show that Mix&Go activated magnetic particles
demonstrate higher antibody binding capacity and yield than commercially available particles; and
increased target yield and purity, without interfering IgG bands in IP, as is the case with Protein G.
Silver Stain
!1!!2!!3!!4!!!5!!!6!!7!
Western Blot
!1!!2!!3!!4!!!5!!!6!!7!
Aim – Multiplex Immunoassay Using the AMG Activation Kit
The differences in methods of antibody attachment between ELISA microtitre plate (passive adsorbtion)
and microspheres (covalent chemistry) often leads to poor immunoassay transfer from the ELISA
platform. The aim is to use antibodies recommended for ELISA and easily couple them to Luminex®
MagPlex® microspheres using the AMG Activation Kit for Multiplex Microspheres. These microspheres
can then be used in a multiplex immunoassay.
Method – Activation Kit
The AMG Activation Kit for
Multiplex Microspheres comes with
activation reagent and buffers
required to activate and couple
antibody to multiplex
microspheres, such as MagPlex®
microspheres for the Luminex®
platform. MagPlex ® microspheres were
coupled with 4 cytokine specific
antibodies, IL-6, TNF, IFN-γ and
GM-CSF (BD Biosciences), using
the included instructions for use.
For comparison TNF was also
coupled to Luminex® microspheres
using the Bio-Rad Bio-Plex™
Amine Coupling Kit and the
Luminex® xMAP® Antibody
Coupling Kit.
Multiplex sandwich immunoassays
were run using known standards
and controls.
xMAP® Antibody
Coupling Kit
AMG Activation Kit
Bio-Plex™ Coupling Kit
6 Steps
8 Steps
120 Minutes 140 Minutes +
O/N
12 Steps
170 Minutes
Prepare EDC (50 mg/mL)
Prepare S-NHS (50mg/mL)
Prepare EDC (40 mg/mL)
Prepare S-NHS (50mg/mL)
Add Mix&Go to Microspheres
(100 µL)
Add EDC and S-NHS to Microspheres
Add EDC and S-NHS to
Microspheres
Incubate 15 – 60 minutes
Incubate 20 minutes
Incubate 20 minutes
Wash Microspheres (150 µL)
Wash Microspheres
(3x 500 µL)
Add Antibody (500 µL)
Wash Microspheres (2x 100 µL)
Add Antibody (100 µL)
Incubate 2 hours
Add Antibody
(500 µL)
Wash Microspheres (500 µL) Incubate 2 hours
Add Blocking Buffer (250 µL)
Incubate 15 – 60 minutes
Wash Microspheres (2x 100 µL) Incubate 30 minutes
Wash Microspheres
(3x 500 µL) Wash Microspheres (500 µL)
Store Microspheres Overnight
Store Microspheres (100 µL)
Store Microspheres (150 µL)
(1,000 µL)
Results – Activation Kit
Heavy Chain
Target
A multiplex sandwich immunoassay was run on a Luminex® 100 system. The Bio-Plex Manager™
Software was used to generate the standard curve. The values for each standard are entered and then 5
place regression chosen. Controls were calculated by the software and plotted against the expected
values in Microsoft® Excel.
Light Chain
A. Coupling Kit Comparison
B. Cytokine Multiplex Standard Curve
C. IL-6
30,000
1,000
25,000
Observed pg/mL
10,000
MFI
MFI
20,000
15,000
1,000
10,000
100
0
10
0
100
200
300
400
500
600
1
10
100
TNF Antigen pg/mL
AMG Activation Kit
25
Bio-Plex
IL-6
TNF
GM-CSF
y = 0.9162x + 11.516
R² = 0.99888
600
Expected pg/mL
800
1000
Observed pg/mL
800
400
600
400
200
y = 1.0374x + 4.0856
R² = 0.99996
0
0
200
400
800
1000
F. TNF
800
600
600
IFN-gamma
800
400
400
Expected pg/mL
1,000
200
200
E. GM-CSF
Observed pg/mL
Observed pg/mL
0
1,000
0
y = 0.8992x + 3.5362
R² = 0.99998
0
1,000
1,000
0
600
Expected pg/mL
800
1000
600
400
200
y = 0.8793x - 1.7073
R² = 0.99987
0
0
200
400
600
800
1000
Expected pg/mL
Figure 5. Graph A shows a comparison of two coupling kits to the AMG Activation Kit using a singleplex TNF sandwich
assay. This shows equivalent signal across all kits. B shows the standard curve of the 4-plex cytokine immunoassay. This
displays good dynamic range and low end signal. This standard curve was used to calculate the observed values.
Observed versus Expected values for the controls were plotted and linear regression performed. This is shown in graphs C
– E, very good correlation (R2 value) and accuracy (slope) was noted and is equivalent to that observed using the ELISA
method.
20
15
10
Conclusion
5
0
Batch 1
Conclusion
Luminex
200
µg Mouse IgG per mg of Particles
Figure 4. Mouse IgG binding capacity of the AMG Coupling Kit, 1
µm magnetic particles. Coupling batches show good reproducibility
with mouse IgG binding capacity of 23 ± 1 µg mouse IgG per mg of
particles. By comparison, Dynal M-280 Protein G particles have a
binding capacity of 8 µg of human IgG per mg (Life Technologies). 30
400
pg/mL
D. IFN-gamma
Mouse IgG Binding Capacity
AMG Coupling Kit, 1µm Magnetic Particles
600
200
5,000
Figure 3. Silver stain of SDS-PAGE gel and western blot. Lanes are loaded with the following:
1: Molecular weight markers
2: A431 Cell Lysate
3: Immuno-precipitation Lysate Supernatant after particle incubation
4: Mix&Go αErk1/2 mAb Acid Elution Replicate 1
5: Mix&Go αErk1/2 mAb Acid Elution Replicate 2
6: Dynal M-280 Protein G αErk1/2 mAb Acid Elution Replicate 1
7: Dynal M-280 Protein G αErk1/2 mAb Acid Elution Replicate 2
800
Batch 2
Batch 3
Coupling Batches
Mix&Go is a simple universal coupling technology that utilises multi-point avidity metal chelation to bind
surfaces with electron donating potential, thereby acting as molecular velcro. The AMG Coupling Kit, 1 µm Magnetic Particles allows the use of pre-activated particles in many areas of
research. The data presented here shows pull down of a target molecule (ERK) after an antibody was
coupled directly to the particles. The resulting IP shows good target depletion from the sample matrix
and excellent final eluted yield and purity. A major advantage to this approach is that the antibody does
not elute off and interfere with the results, as seen with Protein G particles.
The AMG Activation Kit for Multiplex Microspheres from Anteo Technologies, is simple and easy to use. It
enables researchers to easily develop multiplex immunoassays on the Luminex® system using the same
antibodies used for ELISA. Figure 5A shows a comparison to existing coupling kits for Luminex®
microspheres displaying comparable results. Activation and coupling takes fewer steps therefore taking
less time.
Another advantage to using the AMG Activation kit is the ability to store the activated microspheres for
use at a later time. This is not possible with S-NHS/EDC coupling chemistries as the active esters
hydrolyse in water.
A multiplex cytokine assay was also developed with limited optimisation. High precision and accurate
results are obtained when solving for unknowns using a standard curve. In the three examples shown the
expected vs obtained slope was less than 0.1 from 1 (perfect correlation) with an R2 value > 0.996 (Figure
5). This shows that the immunoassay can be easily transferred from ELISA to Luminex®.