The ever increasing pressures on clinical laboratories to becomemore productive and more cost-effective are forcing scientists tore-evaluate the way they carry out test procedures. Since labouraccounts for approximately 65% of the total cost of producingtest results, the most logical solution to the problem of cost con-trol seems to be the automation of the most commonly per-formed assays, such as immunoassays. However, any newly auto-mated test must generate results that match or are superior tothose obtained with manually performed assays. In addition, amuch higher throughput should be attained with minimalrequirements for specialised hands-on-time. To satisfy such chal-lenging criteria, the design of the automated assay, and in partic-ular the solid phase, is critical.

The type of solid phase employed in automated immunoassays isa fundamental part of the assay design process and may ultimate-ly determine the success of the assay. The solid phase must be easyto use and adaptable to assay development, should have highcapacity, have excellent signal-to-noise ratios, and enable efficientand reproducible immobilisation of antibodies or other ligands.Minimal mixing requirements and rapid, effective washing of thesolid phase are also essential in high throughput systems.

The use of magnetic beads as the solid phase in automatedimmunoassays is well established and gaining popularity. As moresystems are developed and the assays become more complex, beadmanufacturers are looking at ways to improve the properties oftheir beads to meet the growing needs of their customers. DynalBiotech has developed a new one micron magnetic bead platform,Dynabeads MyOne, which has been optimised for use as the solidphase in automated immunoassays. Both carboxylic acid andtosylactivated forms are available. The small, uniform size of thebeads provides a large surface area to which antibodies or otherligands can bind, while the carefully controlled iron contentensures that the beads are truly superparamagnetic. This meansthat there is no remanence (magnetism remaining) after remov-ing the magnetic field, so preventing clumping in automated sys-tems. The unique properties of the new beads have been carefully

engineered to provide optimum results in automated immunoas-says, described in more detail below.

Bead size and size distributionTo produce accurate and reproducible results in automatedimmunoassays, all beads used as the solid phase must be identical.Coulter Counter measurements can be performed to determinebead size and size distribution. Figure 1 shows the size distribu-tion for one batch of tosylactivated beads measured with theMultisizer 3 coulter counter (Beckman Coulter, Inc., USA). It canbe seen from the main peak that size variation within this batch isminimal. Many alternative magnetic particles from various sup-pliers have been tested in a similar way and their sizes, both with-in and between batches, were found to vary considerably. As aconsequence, such particles are more likely to give results whichcan not be reproduced when used on automated systems.

The number of tosylactivated beads per gram dry weight was alsodetermined using Coulter Counter measurements and was foundto be approximately 1012 beads per gram dry weight. This meas-

One micron magnetic beadsoptimised for automatedimmunoassays

The use of magnetic beads as the solid phase in automated immunoassays resultsin faster reactions and thus shorter assay times, as well as increased sensitivity.This article describes how the utilisation of a new one micron bead platformallows a larger surface area for protein immobilisation. Fewer beads can be usedwithout compromising either the accuracy of results or the dynamic range. The improved dispersion properties of the new beads, combined with their lowsedimentation rate, removes the need for mixing of reagents.

by Jack Andreassen

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Figure 1. Size distribution of one batch of the tosylactivated beads.

urement is dependent on bead diameter since the volume of thebead is proportional to the cube of the radius.

Magnetic propertiesThe magnetic properties of beads used as the solid phase inautomated systems are clearly of great importance, both duringthe manufacture of the immunoassay and in its application. Toensure that the beads are collected efficiently on the magnet theiron oxide content must be high. However, total resuspension ofthe pellet during washing steps is equally important as rapid,effective washing steps reduce assay time and therefore increaseoverall throughput. To satisfy these requirements the beadsmust be truly superparamagnetic and there should be no rema-nence after removing the magnetic field.

The iron oxide content of Dynabeads MyOne beads is 37%. Themagnetic material is evenly distributed throughout the beads asnanosized iron oxide crystals. This results in superparamagnet-ic behaviour, as shown by the hysteresis curve in Figure 2, wherethe magnetisation is nearly identical with both increasing anddecreasing magnetic fields. The beads show no remanencewhen the magnetic field is zero.

Surface characteristicsHydrophobicity and the charge on the bead surface are impor-tant parameters when coating beads with antibodies or similarproteins generally needed in immunoassays. The initial driving

force when immobilising antibodies on hydrophobic beads,such as the tosylactivated beads, is hydrophobic adsorption tothe bead surface. The chemical covalent bonds between thetosylactivated groups on the bead surface and the protein areonly created after this initial contact. For hydrophilic bead sur-faces the initial contact between the antibody and the surfaceof the bead is electrostatic or via random interactions, or itmay occur following activation of functional groups on thebead surface.

The degree of hydrophobicity of the tosylactivated beads as wellas the carboxylic acid version of the beads was determined bymeasuring the contact angle of water droplets deposited on alayer of dry beads using a Fibro Dat 1120 instrument (Thwing-Albert Instrument Company, USA). The higher the contactangle observed, the more hydrophobic the bead surface.

The isoelectric point for each of the bead types was also deter-mined using the Zetasizer 2000-3000 HS (MalvernInstruments, UK) to measure the Zeta potential. These meas-urements were performed on uncoated beads as well as onbeads coated with antibody or streptavidin, to assess whetherthe surface properties of the beads are affected by the immo-bilised protein. The results are shown in Table 1.

The hydrophobicity of the tosylactivated beads is very compa-rable, whereas the carboxylic acid beads are, in contrast, very

hydrophilic due to their high negativenet charge at neutral pH. Zeta potentialmeasurements were performed on threedifferent types of beads (two sizes oftosylactivated beads and carboxylic acidbeads) after coating with monoclonalmouse IgG1 antibody and streptavidin[Table 1]. These results show that theisoelectric point of the beads is princi-pally determined by the nature of theimmobilised protein. For example, aftercoating the originally negatively chargedcarboxylic acid beads with antibody,their isoelectric point is similar to themore neutral tosylactivated beads coat-ed with the same antibody.

Figure 2. Hysteresis curve for the bead platform: magnetisation as a func-tion of magnetic field strength. The magnetisation curves are overlappingboth when increasing and decreasing the magnetic field, and the beadsshow superparamagnetic behaviour.

Figure 3. Sedimentation of 1.0 µm and 2.8 µm beads in aqueous solu-tion measured as relative absorbance at 450 nm as a function of settlingtime (minutes).

Table 1. Relative contact angle and isoelectric point measured with Zeta potential.

I mmunodiagnostics As Published in CLI April 2005

Understanding the surface characteristics of the different typesof beads is important in immunoassay development in order toestablish the optimal binding kinetics for both the ligand bind-ing to the bead surface, as well as for the target protein bindingto the immobilised ligand. Electrostatic interactions (repulsionforces) may lower the binding efficiency, particularly if the sur-face of the beads is negatively charged.

Antibody immobilisationThe immobilisation efficiency of the coating of magnetic beadswith proteins such as antibodies or streptavidin, is very importantsince it affects the number of beads required in the solid phase aswell as the dynamic range that can be achieved by the assay. Therate of immobilisation depends on the properties of the protein,such as its size and isoelectric point, as well as the relative concen-trations of the protein and magnetic beads. The nature of thebuffer used during the immobilisation is also important. To meas-ure the efficiency of antibody immobilisation to the tosylactivatedbeads, I125-labelled antibody was added to the beads. It was foundthat, depending on the antibody type, 60-95% of the antibody wasbound to the beads following incubation. The degree of covalentbinding was then measured using SDS to remove physicallyadsorbed protein. It was found that 95-100% of the antibodiesbound to the tosylactivated beads were linked covalently, thusreducing the risk of protein leakage from the beads during storage.

SedimentationIn automated immunoassays the opportunities for mixing duringincubation and washing steps may be limited. It is thereforeimportant for beads to have a slow sedimentation rate to preventthem from settling at the bottom of the cuvette. Both the size anddensity of the beads determine the rate of sedimentation, whichcan be measured using a spectrophotometer as the beads areallowed to settle over time. As the beads settle the solution becomesless dense optically, and consequently the absorbance reading at450 nm decreases. The sedimentation rate for 2.8 µm streptavidinbeads and 1.0 µm streptavidin beads were compared over time andthe results are shown in Figure 3. Both the2.8 µm and the 1.0 µm beads remained insuspension for the first 30 minutes. Whilethe 2.8 µm beads almost completely settledafter 100 minutes, the 1.0 µm beads werestill in solution after 3 hours. It can be con-cluded, therefore, that using the 1.0 µmstreptavidin beads in automatedimmunoassays eliminates the need formixing during the incubation steps.

Titration of the beads in immunoassaysThe number of beads required in the solidphase of an immunoassay may be deter-mined by the need for a wide dynamicrange. In addition to parameters such asimmobilisation efficiency, the size of thebeads is also important since smaller beadshave a larger surface area available forimmobilisation. As the geometrical surfacearea per weight for 1.0 µm beads is approx-imately 2.5 times higher than 2.8 µmbeads, theoretically fewer 1.0 µm beadsshould be required to achieve the sameaccuracy in an assay.

To determine whether this is the case, 2.8 µm tosylactivated beadsand 1µm tosylactivated beads were coated with antibody againstD-dimer in a model system and used in a sandwich immunoas-say for D-dimer on an automated random access bench topanalyser. As a reference, the required weight of the 2.8 µm tosy-lactivated beads was taken to be 100%. Preparations containing30, 40 and 50% weight of the 1µm tosylactivated beads were thenused in the assay and the results compared to those obtained withthe 2.8 µm beads Figure 4. The results indicate that the increasedsurface area of the 1.0 µm beads reduced the weight of beadsrequired in the assay by 60%. This may be used to increase thedynamic range of the assay.

This conclusion that a reduced weight of 1.0 µm beads is neededcompared to 2.8 µm beads is also valid with other assays. A num-ber of further experiments were performed. Both bead sizes werecoated with capture antibodies for D-dimer and myoglobulin foruse in a sandwich immunoassay on a automated random accessbench top analyser. Two preparations of different sizes of strep-tavidin-coated beads were also used in a sandwich immunoassayfor intact parathyroid hormone (PTH) utilising a biotinylated

Figure 4. Bead titration of different amounts of 1.0 µm beads in a D-dimer immunoassay compared with 2.8 µm beads..

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

capture antibody as a model system. The results, shown in Figures5, 6 and 7, confirm that the amount of 1µm beads required in allassays is just 40% of the weight required of 2.8 µm beads.

ReproducibilityWhen manufacturing assay components such as magnetic beadscoated with antibodies or similar proteins, reproducible resultsare essential. All batches of the 1µm beads are validated at the pro-duction scale. Validation batches are then further analysed usingstandard QC methods to ensure that the physical and chemicalproperties of the beads in each batch are identical. Three differentbatches of the tosylactivated beads were coated with streptavidinand tested in an immunoassay for PTH utilising a biotinylatedcapture antibody. Experimental batches of beads with lowerbiotin binding capacities were also included to determine thethreshold biotin binding capacity required to achieve repro-ducible results in the PTH assay. The results, shown in Figure 8,demonstrate the excellent reproducibility of the 1.0 µm beadmanufacturing processes, since the validation batches (shown inred) compare very well with the process qualification batch

(shown in purple). The low-bindingexperimental batches are shown in blue.Similarly, the validation batches werecoated directly with PTH antibody andtested in a standard sandwichimmunoassay. All four batches of tosylac-tivated beads behaved identically in thePTH assay, demonstrating the excellentreproducibility of the antibody immobil-isation process used to coat the tosylacti-vated beads.

ConclusionDynabeads MyOne beads have been care-fully engineered to create an optimummagnetic solid phase for automatedimmunoassays. Their small, uniform sizeprovides a large surface area for consis-tent protein immobilisation and enables60% less weight of beads to be used inassays without compromising the accura-cy or the dynamic range. Good dispersionproperties combined with a slow sedi-mentation rate remove the need for stir-

ring during incubation and washing steps, while the high ironoxide content ensures excellent separation properties with noremanence. The beads can be used to improve the dynamic rangeand signal-to-noise ratio of existing immunoassays with greatlyreduced need for assay optimisation.

The authorJack Andreassen, M.Sc.,Senior International Product Manager,IVD / OEM SupportDynal Biotech,Ullernchausseen 52N-0309,Oslo,NorwayThe work was carried out in collaboration with Future Diagnostics, bv.

Figure 8. Response units in a sandwich PTH assay employing streptavidin coated beads and abiotinylated capture antibody as a function of the measured free biotin binding capacity of the strep-tavidin coated beads. The validation batches (red) compare very well with the process qualificationbatch (purple). Experimental batches with different biotin binding capacities are depicted in blue.

Figure 7. Standard curve measurement for intact PTH immunoassaywith 2.8 µm beads (100 % weight), and 1µm beads (40 % weight).

Figure 6. Standard curve measurement for Myoglobulin immunoassaywith 2.8 µm beads (100 % weight), and 1µm beads (40 % weight).

I mmunodiagnostics As Published in CLI April 2005