Basic Principles of Magnetic Affinity Separations

In general, magnetic affinity separations can be performed in two different modes. In the direct method an appropriate affinity ligand is directly coupled to the magnetic particles or biopolymer exhibiting affinity towards target compound(s) is used in the course of preparation of magnetic affinity particles. These particles are added to the sample and target compounds then bind to them. In the indirect method the free affinity ligand (in most cases an appropriate antibody) is added to the solution or suspension to enable the interaction with the target compound. The resulting complex is then captured by appropriate

Figure 1 (See Colour Plate 61). Examples of test-tube magnetic separators (Dynal, Norway). Left, Dynal MPC-6; right, Dynal MPC-1. Courtesy of Dynal, Oslo, Norway.
Figure 2 (See Colour Plate 62). A typical example of laboratory-scale high-gradient magnetic separators. OctoMACS Separator (Miltenyi Biotec, Germany) can be used for simultaneous isolation of mRNA. Courtesy of Miltenyi Biotec, Germany.

magnetic particles. In case antibodies are used as free affinity ligands, magnetic particles with immobilized secondary antibodies, protein A or protein G are used for capturing the complex. Alternatively, the free affinity ligands can be biotinylated and magnetic particles with immobilized streptavidin or avidin are used to capture the complexes formed. In both methods magnetic particles with isolated target com-pound(s) are magnetically separated and then a series of washing steps are performed to remove the majority of contaminating compounds and particles. The target compound is then usually eluted, but for specific applications (especially in molecular biology, bi-oanalytical chemistry or environmental chemistry) they can be used still attached to the particles, such as in the case of polymerase chain reaction, magnetic ELISA, etc.

The two methods perform equally well, but, in general, the direct technique is more easily controlled. The indirect procedure may perform better if affinity ligands have poor affinity for the target compound.

In some cases, nonspecific binding of accompanying compounds can be observed due to the specific properties of the magnetic particle material. In this case, pretreatment with the magnetic carrier without immobilized affinity ligand or with immobilized nonspecific molecules will usually help. The nonspecific binding can be also minimized by adding a nonionic detergent both in the sample and in the washing buffers after isolation of the target.

In most cases, magnetic batch affinity adsorption is used to perform the separation step. This approach represents the simplest procedure available, enabling the whole separation to be performed in one test-tube or flask. If larger magnetic particles (with diameters ca. > 1 |im) are used, simple magnetic separators can be employed. If magnetic colloids (diameters ranging between tens and hundreds of nanometers) are used as affinity adsorbents, high-gradient magnetic separators have usually to be used to remove the magnetic particles from the system.

Alternatively, magnetically stabilized fluidized beds (MSFB), which allow continuous separation, can be used. The use of MSFB is an alternative to conventional column operation, such as packed bed or fluidized bed, especially for large-scale purification of biological products. Magnetic stabilization enables the expansion of a packed bed without mixing of solid particles. High column efficiency, low pressure drop and elimination of clogging can be attained.

Biocompatible two-phase systems, composed for example from dextran and polyethylene glycol, are often used for isolation of biologically active compounds, subcellular organelles and cells. The separation of the phases can be accelerated by the addition of fine magnetic particles or ferrofluids to the system followed by the application of a magnetic field. Magnetically enhanced phase separation usually increases the speed of phase separation by a factor of about 10 in easy systems, but it may increase by a factor of many thousands in difficult systems.

Solar Panel Basics

Solar Panel Basics

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