One micron magnetic beads optimised for automated immunoassays

lab technology
I mmunodiagnostics
One micron magnetic beads
optimised for automated
immunoassays
by Jack Andreassen
As Published in CLI April 2005
The use of magnetic beads as the solid phase in automated immunoassays results
in faster reactions and thus shorter assay times, as well as increased sensitivity.
This article describes how the utilisation of a new one micron bead platform
allows a larger surface area for protein immobilisation. Fewer beads can be used
without compromising either the accuracy of results or the dynamic range.
The improved dispersion properties of the new beads, combined with their low
sedimentation rate, removes the need for mixing of reagents.
The ever increasing pressures on clinical laboratories to become
more productive and more cost-effective are forcing scientists to
re-evaluate the way they carry out test procedures. Since labour
accounts for approximately 65% of the total cost of producing
test results, the most logical solution to the problem of cost control
seems to be the automation of the most commonly performed
assays, such as immunoassays. However, any newly automated
test must generate results that match or are superior to
those obtained with manually performed assays. In addition, a
much higher throughput should be attained with minimal
requirements for specialised hands-on-time. To satisfy such challenging
criteria, the design of the automated assay, and in particular
the solid phase, is critical.
The type of solid phase employed in automated immunoassays is
a fundamental part of the assay design process and may ultimately
determine the success of the assay. The solid phase must be easy
to use and adaptable to assay development, should have high
capacity, have excellent signal-to-noise ratios, and enable efficient
and reproducible immobilisation of antibodies or other ligands.
Minimal mixing requirements and rapid, effective washing of the
solid phase are also essential in high throughput systems.
The use of magnetic beads as the solid phase in automated
immunoassays is well established and gaining popularity. As more
systems are developed and the assays become more complex, bead
manufacturers are looking at ways to improve the properties of
their beads to meet the growing needs of their customers. Dynal
Biotech has developed a new one micron magnetic bead platform,
Dynabeads MyOne, which has been optimised for use as the solid
phase in automated immunoassays. Both carboxylic acid and
tosylactivated forms are available. The small, uniform size of the
beads provides a large surface area to which antibodies or other
ligands can bind, while the carefully controlled iron content
ensures that the beads are truly superparamagnetic. This means
that there is no remanence (magnetism remaining) after removing
the magnetic field, so preventing clumping in automated systems.
The unique properties of the new beads have been carefully
engineered to provide optimum results in automated immunoassays,
described in more detail below.
Bead size and size distribution
To produce accurate and reproducible results in automated
immunoassays, all beads used as the solid phase must be identical.
Coulter Counter measurements can be performed to determine
bead size and size distribution. Figure 1 shows the size distribution
for one batch of tosylactivated beads measured with the
Multisizer 3 coulter counter (Beckman Coulter, Inc., USA). It can
be seen from the main peak that size variation within this batch is
minimal. Many alternative magnetic particles from various suppliers
have been tested in a similar way and their sizes, both within
and between batches, were found to vary considerably. As a
consequence, such particles are more likely to give results which
can not be reproduced when used on automated systems.
The number of tosylactivated beads per gram dry weight was also
determined using Coulter Counter measurements and was found
to be approximately 10 12 beads per gram dry weight. This meas-
Figure 1. Size distribution of one batch of the tosylactivated beads.

I mmunodiagnostics
As Published in CLI April 2005
Figure 2. Hysteresis curve for the bead platform: magnetisation as a function
of magnetic field strength. The magnetisation curves are overlapping
both when increasing and decreasing the magnetic field, and the beads
show superparamagnetic behaviour.
Figure 3. Sedimentation of 1.0 µm and 2.8 µm beads in aqueous solution
measured as relative absorbance at 450 nm as a function of settling
time (minutes).
urement is dependent on bead diameter since the volume of the
bead is proportional to the cube of the radius.
Magnetic properties
The magnetic properties of beads used as the solid phase in
automated systems are clearly of great importance, both during
the manufacture of the immunoassay and in its application. To
ensure that the beads are collected efficiently on the magnet the
iron oxide content must be high. However, total resuspension of
the pellet during washing steps is equally important as rapid,
effective washing steps reduce assay time and therefore increase
overall throughput. To satisfy these requirements the beads
must be truly superparamagnetic and there should be no remanence
after removing the magnetic field.
The iron oxide content of Dynabeads MyOne beads is 37%. The
magnetic material is evenly distributed throughout the beads as
nanosized iron oxide crystals. This results in superparamagnetic
behaviour, as shown by the hysteresis curve in Figure 2, where
the magnetisation is nearly identical with both increasing and
decreasing magnetic fields. The beads show no remanence
when the magnetic field is zero.
Surface characteristics
Hydrophobicity and the charge on the bead surface are important
parameters when coating beads with antibodies or similar
proteins generally needed in immunoassays. The initial driving
Table 1. Relative contact angle and isoelectric point measured with Zeta potential.
force when immobilising antibodies on hydrophobic beads,
such as the tosylactivated beads, is hydrophobic adsorption to
the bead surface. The chemical covalent bonds between the
tosylactivated groups on the bead surface and the protein are
only created after this initial contact. For hydrophilic bead surfaces
the initial contact between the antibody and the surface
of the bead is electrostatic or via random interactions, or it
may occur following activation of functional groups on the
bead surface.
The degree of hydrophobicity of the tosylactivated beads as well
as the carboxylic acid version of the beads was determined by
measuring the contact angle of water droplets deposited on a
layer of dry beads using a Fibro Dat 1120 instrument (Thwing-
Albert Instrument Company, USA). The higher the contact
angle observed, the more hydrophobic the bead surface.
The isoelectric point for each of the bead types was also determined
using the Zetasizer 2000-3000 HS (Malvern
Instruments, UK) to measure the Zeta potential. These measurements
were performed on uncoated beads as well as on
beads coated with antibody or streptavidin, to assess whether
the surface properties of the beads are affected by the immobilised
protein. The results are shown in Table 1.
The hydrophobicity of the tosylactivated beads is very comparable,
whereas the carboxylic acid beads are, in contrast, very
hydrophilic due to their high negative
net charge at neutral pH. Zeta potential
measurements were performed on three
different types of beads (two sizes of
tosylactivated beads and carboxylic acid
beads) after coating with monoclonal
mouse IgG1 antibody and streptavidin
[Table 1]. These results show that the
isoelectric point of the beads is principally
determined by the nature of the
immobilised protein. For example, after
coating the originally negatively charged
carboxylic acid beads with antibody,
their isoelectric point is similar to the
more neutral tosylactivated beads coated
with the same antibody.

I mmunodiagnostics
As Published in CLI April 2005
Understanding the surface characteristics of the different types
of beads is important in immunoassay development in order to
establish the optimal binding kinetics for both the ligand binding
to the bead surface, as well as for the target protein binding
to the immobilised ligand. Electrostatic interactions (repulsion
forces) may lower the binding efficiency, particularly if the surface
of the beads is negatively charged.
Antibody immobilisation
The immobilisation efficiency of the coating of magnetic beads
with proteins such as antibodies or streptavidin, is very important
since it affects the number of beads required in the solid phase as
well as the dynamic range that can be achieved by the assay. The
rate of immobilisation depends on the properties of the protein,
such as its size and isoelectric point, as well as the relative concentrations
of the protein and magnetic beads. The nature of the
buffer used during the immobilisation is also important. To measure
the efficiency of antibody immobilisation to the tosylactivated
beads, I 125 -labelled antibody was added to the beads. It was found
that, depending on the antibody type, 60-95% of the antibody was
bound to the beads following incubation. The degree of covalent
binding was then measured using SDS to remove physically
adsorbed protein. It was found that 95-100% of the antibodies
bound to the tosylactivated beads were linked covalently, thus
reducing the risk of protein leakage from the beads during storage.
Sedimentation
In automated immunoassays the opportunities for mixing during
incubation and washing steps may be limited. It is therefore
important for beads to have a slow sedimentation rate to prevent
them from settling at the bottom of the cuvette. Both the size and
density of the beads determine the rate of sedimentation, which
can be measured using a spectrophotometer as the beads are
allowed to settle over time. As the beads settle the solution becomes
less dense optically, and consequently the absorbance reading at
450 nm decreases. The sedimentation rate for 2.8 µm streptavidin
beads and 1.0 µm streptavidin beads were compared over time and
the results are shown in Figure 3. Both the
2.8 µm and the 1.0 µm beads remained in
suspension for the first 30 minutes. While
the 2.8 µm beads almost completely settled
after 100 minutes, the 1.0 µm beads were
still in solution after 3 hours. It can be concluded,
therefore, that using the 1.0 µm
streptavidin beads in automated
immunoassays eliminates the need for
mixing during the incubation steps.
Figure 4. Bead titration of different amounts of 1.0 µm beads in a D-
dimer immunoassay compared with 2.8 µm beads..
To determine whether this is the case, 2.8 µm tosylactivated beads
and 1µm tosylactivated beads were coated with antibody against
D-dimer in a model system and used in a sandwich immunoassay
for D-dimer on an automated random access bench top
analyser. As a reference, the required weight of the 2.8 µm tosylactivated
beads was taken to be 100%. Preparations containing
30, 40 and 50% weight of the 1µm tosylactivated beads were then
used in the assay and the results compared to those obtained with
the 2.8 µm beads Figure 4. The results indicate that the increased
surface area of the 1.0 µm beads reduced the weight of beads
required in the assay by 60%. This may be used to increase the
dynamic range of the assay.
This conclusion that a reduced weight of 1.0 µm beads is needed
compared to 2.8 µm beads is also valid with other assays. A number
of further experiments were performed. Both bead sizes were
coated with capture antibodies for D-dimer and myoglobulin for
use in a sandwich immunoassay on a automated random access
bench top analyser. Two preparations of different sizes of streptavidin-coated
beads were also used in a sandwich immunoassay
for intact parathyroid hormone (PTH) utilising a biotinylated
Titration of the beads in immunoassays
The number of beads required in the solid
phase of an immunoassay may be determined
by the need for a wide dynamic
range. In addition to parameters such as
immobilisation efficiency, the size of the
beads is also important since smaller beads
have a larger surface area available for
immobilisation. As the geometrical surface
area per weight for 1.0 µm beads is approximately
2.5 times higher than 2.8 µm
beads, theoretically fewer 1.0 µm beads
should be required to achieve the same
accuracy in an assay.
Figure 5. Standard curve measurement for D-dimer immunoassay with 2.8 µm beads (100 %
weight), and 1µm beads (40 % weight).

I mmunodiagnostics
As Published in CLI April 2005
Figure 6. Standard curve measurement for Myoglobulin immunoassay
with 2.8 µm beads (100 % weight), and 1µm beads (40 % weight).
Figure 7. Standard curve measurement for intact PTH immunoassay
with 2.8 µm beads (100 % weight), and 1µm beads (40 % weight).
Figure 8. Response units in a sandwich PTH assay employing streptavidin coated beads and a
biotinylated capture antibody as a function of the measured free biotin binding capacity of the streptavidin
coated beads. The validation batches (red) compare very well with the process qualification
batch (purple). Experimental batches with different biotin binding capacities are depicted in blue.
capture antibody as a model system. The results, shown in Figures
5, 6 and 7, confirm that the amount of 1µm beads required in all
assays is just 40% of the weight required of 2.8 µm beads.
Reproducibility
When manufacturing assay components such as magnetic beads
coated with antibodies or similar proteins, reproducible results
are essential. All batches of the 1µm beads are validated at the production
scale. Validation batches are then further analysed using
standard QC methods to ensure that the physical and chemical
properties of the beads in each batch are identical. Three different
batches of the tosylactivated beads were coated with streptavidin
and tested in an immunoassay for PTH utilising a biotinylated
capture antibody. Experimental batches of beads with lower
biotin binding capacities were also included to determine the
threshold biotin binding capacity required to achieve reproducible
results in the PTH assay. The results, shown in Figure 8,
demonstrate the excellent reproducibility of the 1.0 µm bead
manufacturing processes, since the validation batches (shown in
red) compare very well with the process qualification batch
(shown in purple). The low-binding
experimental batches are shown in blue.
Similarly, the validation batches were
coated directly with PTH antibody and
tested in a standard sandwich
immunoassay. All four batches of tosylactivated
beads behaved identically in the
PTH assay, demonstrating the excellent
reproducibility of the antibody immobilisation
process used to coat the tosylactivated
beads.
Conclusion
Dynabeads MyOne beads have been carefully
engineered to create an optimum
magnetic solid phase for automated
immunoassays. Their small, uniform size
provides a large surface area for consistent
protein immobilisation and enables
60% less weight of beads to be used in
assays without compromising the accuracy
or the dynamic range. Good dispersion
properties combined with a slow sedimentation
rate remove the need for stirring
during incubation and washing steps, while the high iron
oxide content ensures excellent separation properties with no
remanence. The beads can be used to improve the dynamic range
and signal-to-noise ratio of existing immunoassays with greatly
reduced need for assay optimisation.
The author
Jack Andreassen, M.Sc.,
Senior International Product Manager,
IVD / OEM Support
Dynal Biotech,
Ullernchausseen 52N-0309,
Oslo,
Norway
The work was carried out in collaboration with
Future Diagnostics, bv.