Examples of Magnetic Affinity Separations of Biologically Active Compounds and Xenobiotics

Magnetic affinity separations have been successfully used in various areas, such as molecular biology, biochemistry, immunochemistry, enzymology, analytical chemistry and environmental chemistry. Tables 2-4 show some selected applications of these techniques.

At present, magnetic affinity separation techniques are used especially in molecular biology for the isolation of RNA, DNA and oligonucleotides. Almost all the procedures employ the same basic principle, based on the hybridization of immobilized oligo-nucleotides and target structures. There are several companies offering oligodeoxythymidine immobilized on magnetic particles, which can be effectively used for rapid isolation of highly purified, intact poly A+mRNA from eukaryotic total RNA. Poly A+ mRNA has been successfully isolated from various samples, such as cells, animal and plant tissues, blood, cells isolated by immunomagnetic separation, paraffin-embedded tissues, etc. Also cells and tissues containing high RNase activities can be used for mRNA isolation. The separated mRNA can be eluted from the beads by lowering the ionic strength of the elution buffer and used for further applications (Northern blotting, dot-blot hybridization, hybridization probes) or used still bound to magnetic particles (cDNA synthesis, construction of solid-phase cDNA library, etc.). Enzymatic downstream applications are usually not inhibited by the presence of magnetic particles. The covalent binding of oligodeo-xythymidine to magnetic particles makes it possible to regenerate the specific adsorbent and to reuse it up to four times.

Isolation of DNA and RNA can be simply performed using biotinylated specific nucleic acids or oligonucleotides immobilized on magnetic particles with immobilized streptavidin. Usually large binding capacity can be achieved resulting in excellent reaction kinetics and high efficiency of the procedure. In addition, magnetic silica particles have been used for simple isolation of DNA and RNA from various biological samples and also to purify DNA fragments after agarose gel electrophoresis.

Table 2 Typical examples of magnetic separations of nucleic acids

Nucleic acid

Magnetic system used

Typical examples

RNA

Magnetic particles with immobilized oligo (dT)25

Eukaryotic poly A# mRNA, viral poly

A # RNA

Magnetic particles with immobilized specific oligonucleotides

tRNA

DNA

Dynabeads DNA DIRECT

PCR-ready DNA

Biotinylated cloned genomic DNA immobilized on Dynabeads

cDNA

Streptavidin

Dynabeads M-280 Streptavidin with immobilized biotinylated

M13 single-stranded DNA

oligonucleotide complementary to the lacZ region

Magnetic particles with immobilized pyrimidine oligonucleotide

Double-stranded target DNA (triple

helix formation)

COOH-terminated magnetic beads (under specific concentrations

Double-stranded DNA, PCR products,

of polyethylene glycol and salt)

M13 single-stranded DNA

Magnetic silica particles

DNA

Table 3 Selected examples of magnetic affinity separation of proteins

Isolated protein Magnetic system used

Typical examples

Enzymes Sub-micron ferrite particles with immobilized soybean trypsin inhibitor

Ferrofluid-modified 5'-AMP-Sepharose 4B Magnetic agarose beads with immobilized dye Magnetic chitin

Dynabeads with immobilized polyclonal antibodies Iminodiacetic acid coupled to magnetic agarose and charged with Zn2 +

Alginate-magnetite beads

Magnetic affinity aqueous two-phase system

Trypsin

Alcohol dehydrogenase Lactate dehydrogenase Lysozyme

Angiotensin-converting enzyme AngioI-TEM-ß-lactamase

Pectinase Hexokinase

Antibodies Magnetic particles with immobilized antibodies against human

Magnetic particles with immobilized protein A or protein G Human serum albumin immobilized onto ferromagnetic dacron

Human IgG antibodies Antibodies

Antibodies against human serum albumin

Lectins Magnetic cross-linked chitosan

Receptors Dynabeads M-450 sheep anti-mouse IgG1 with immobilized monoclonal antibody

Magnetic particles with immobilized oligonucleotide containing EcdR binding sequence

Solanum tuberosum lectin

Human transferrin receptor

Ecdysteroid receptor (EcdR) from Drosophila melanogaster

Other proteins Magnetic particles with immobilized DNA/RNA fragment containing the specific binding sequence Magnetic particles with immobilized m-aminophenylboronic acid Organomercurial-agarose magnetic beads

Ni-NTA Magnetic agarose beads

DNA/RNA binding proteins

Glycated haemoglobin Transcriptionally active chromatin restriction fragments with accessible histone H3 thiols 6xHis-tagged proteins

Table 4 Selected examples of magnetic separation of low-molecular-weight biologically active compounds and organic and inorganic xenobiotics

Type ofcompound

Magnetic system used

Typical examples

Biologically active compounds

Organic xenobiotics

Inorganic xenobiotics

Magnetic particles with immobilized aldosterone antiserum Molecularly imprinted polymer containing magnetic iron oxide Magnetic charcoal

Magnetic particles with immobilized Cu-phthalocyanine

Magnetic polyethyleneimine microcapsules Magnetic particles with immobilized specific antibodies

Bacterial cells adsorbed to magnetite Magnetic charcoal

Magnetic chitosan

Cells of Enterobacter spp. immobilized on magnetite

Magnetic cross-linked cell walls of Saccharomyces cerevisiae Magnetotactic bacteria Solvent extractants on magnetic microparticles

Aldosterone

(S)-propranolol

Separation of free antigens in radioimmunoassays

Polyaromatic hydrocarbons, triphenylmethane dyes

Carcinogens

Pesticides, polyaromatic hydrocarbons, TNT, PCBs

Chlorinated hydrocarbons and pesticides Water-soluble dyes, pesticides

Cu2+ ions Ni2+ ions

Ions of heavy metals

Ions of heavy metals Radionuclides

In the case of protein separation no simple strategy for magnetic affinity separations exists. Various affinity ligands have been immobilized on magnetic particles, or magnetic particles have been prepared from biopolymers exhibiting affinity for target enzymes or lectins, as shown in Table 3. Immunomagnetic particles, i.e. magnetic particles with immobilized specific antibodies against the target structures, have been used for the isolation of various antigens, both molecules and cells and can thus be used for the separation of specific proteins. Enzyme isolation is usually performed using immobilized inhibitors, cofactors, dyes or other suitable ligands, or magnetic beads prepared from affinity biopolymers are used. A general procedure, especially from the point of view of recombinant oligohistidine-tagged proteins, is based on the application of metal chelate magnetic adsorbents. Another general procedure employs immobilized protein A or protein G for the specific separation of immunoglobulins from ascites, serum and tissue culture supernatants.

Magnetic separation of low-molecular-weight biologically active compounds has been used in the course of their determination by various types of immunoassays. Usually immunomagnetic particles directly capture the target analyte, or magnetic particles with immobilized streptavidin are used to capture the complex of biotinylated primary antibody and the analyte. The separated analyte is then determined using an appropriate method.

Isolation and separation of organic and inorganic xenobiotics from environmental and clinical samples using magnetic techniques may find useful applications in the near future. Immobilized copper phthalocyanine dye has an affinity for planar organic compounds, such as polyaromatic hydrocarbons with three and more fused aromatic rings in their molecules, and for triphenylmethane dyes, both groups representing real or potential carcinogens and mutagens. Immunomagnetic separation of xenobiotics such as pesticides, TNT or PCBs is used as a first step in the course of their immunoassay.

Magnetic solid-phase extraction (MSPE) enables preconcentration of target analytes (e.g. environmental contaminants) from large volumes of solutions or suspensions using relatively small amount of magnetic specific adsorbent. This procedure can substitute the standard liquid-liquid and solid-phase extraction procedures.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

Get My Free Ebook


Post a comment