One micron magnetic beads optimised for automated immunoassays

More documents

Recommendations

Info

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).
Page 1: lab technology I mmunodiagnostics O

One micron magnetic beads optimised for automated immunoassays

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?