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Date: June 22, 1998Table 1:Paramagnetic Particles, Table 2:Primary Antibodies Magnetic separations in biology and biotechnology have diversified in recent years, leading to a bewildering array of different particles, affinity mechanisms, and processes. Applications in the nucleic acid realm include products for total and poly(A)+ mRNA isolation from cells or previously purified total RNA preparations, solid-phase cDNA library construction, double-stranded and single-stranded DNA purification, solid phase DNA sequencing, and a variety of hybridization-based methodologies. Magnetic beads also are finding uses in protein purification, immunology, and the isolation of a wide range of specific mammalian cells, bacteria, viruses, subcellular organelles, and individual proteins. There are also products that employ magnetic particles for more conventional isolation and purification methods such as affinity and ion exchange and charcoal trapping of small analytes. It is difficult to know where to start! Fortunately, with few exceptions, most magnetic particles fall into three broad classes: unmodified (or "naked") particles, chemically derivatized particles with general specificity ligands, and chemically derivatized particles with specific recognition groups. General specificity particles are largely produced as substrates to attach a variety of affinity ligands (beads with specific chemical surface modifications allow a broad range of direct applications). Beads are available with a variety of ligands, such as oligo-(dT), streptavidin, or protein-A, attached. Beads can also be coated with highly specific recognition groups (polyclonal and monoclonal antibodies) for very specialized applications. Of course, some products fall in-between these generalizations, or are completely outside of this classification, but we will discuss many products within this scheme. It will be helpful to look at some universal ideas and definitions. Magnetic separations work due to an affinity group on the surface of the magnetic particle. A suspension of these particles is thoroughly mixed with a preparation of the target molecule or cell. After an incubation period, during which the target binds to the affinity ligand, a powerful magnet is used to immobilize the magnetic particles and their trapped analytes (analogous to a centrifugation step in many protocols but more amenable to automation. The simplicity of the magnetic separation has resulted in much recent interest). The unbound material can be removed by aspiration and the bound material washed and detected. Several protocols incorporate methods to detach the trapped analyte from the bead. Magnetic separations subject analytes to very little mechanical stress compared to other methods, they are rapid, often highly scalable, low cost, and they avoid hazardous or toxic reagents. Although often referred to as magnetic, most of the particles currently used are, in fact, paramagnetic. If the magnetic components (generally an iron derivative) are small enough, they will respond to a magnetic field but are incapable of becoming independently magnetic. This is important, as it results in particles that are attracted to a magnetic field yet lose all attraction for each other in the absence of a magnetic field--allowing efficient separation and complete resuspension. DYNAL'S Dynabead M-280 Table 1 shows a selection of companies and some of the magnetic particles they produce, roughly divided into uncoated/chemically terminated, coated with ligands for general specificity, and coated with highly specific ligands. Many of the paramagnetic particles discussed here are in the 1 µm to 5 µm range. In general, what is listed as a 1µm particle will actually cover particles having a small range of sizes, from approximately 0.5 µm to 1.5 µm. Similar ranges are also implied for the larger particles. Monodisperse particles have tightly controlled size ranges (for example, DYNAL's M-450 Dynabeads have an average size of 4.5 µm +/- 0.2 µm) while polydisperse particles have a greater size distribution (CORTEX BIOCHEM's MagaCell particles cover from ~1 µm to ~10 µm, with 80 percent of the particles falling in the 2 µm -4 µm range). There also are differences in the distribution of the magnetic material throughout the particle. Many of the first magnetizable particles, dating in some cases to the early seventies, were micron-sized lumps of iron oxide that were coated with derivatized silane and later with polymer. While many beads still have this basic structure (a coated paramagnetic core) there are alternatives. CORTEX BIOCHEM's particles (e.g. MagaCell) are made by mixing fine Fe3O powder with a polymer, and then grinding and sieving the aggregate, but their MagaBeads have the Fe3O evenly incorporated into the beads during emulsion polymerization. DYNAL's Dynabeads product also has an even dispersion of magnetic material (Fe2O3 and Fe3O4) throughout their 2.8 µm, 4.5 µm, and 5 µm beads, while Spherotech's magnetic particles (1 µm, 2.5 µm, 4 µm, and 7 µm) have a polystyrene core with an iron oxide/polystyrene coating. For many applications, the exact construction of the particle will not matter; however, Bangs Laboratories' Mary Meza cautioned that surface iron can sometimes be a problem. She explained that "people who use some polymerases find that exposure to iron decreases the enzyme's activity, and many cell types are also adversely affected by iron exposure." If exposed iron could be a problem, a particle such as Bangs Laboratories' estapor® Encapsulated microspheres, DYNAL's Dynabeads, or Spherotech's smooth surface magnetic particles (all of which have an outer layer of pure polymer) will be safer. For several years, DYNAL (Norway) has had a significant presence in the biological magnetic particle market. Product manager Rosemary Trovato said "We're specialists. Dynabeads and biomagnetic separations are the backbone of our entire bioscience product line. That's what we do--we make beads." DYNAL's focus on this technology has definitely paid off. Their catalog claims over 2000 references for its cellular isolation products alone. Of the references identified from a brief 1997 MedLine search, the majority of those that explicitly identified a bead used some form of Dynabeads. But many other products are available; some perform purifications that are outside of DYNAL's repertoire, and others are adequate but less expensive. Table 1 describes the scope of products marketed by a number of companies, and the following sections will describe some of these products in more detail. Regardless of how the particles or beads are synthesized, their surfaces will have some form of characteristic chemistry. This section covers particles that are intended to be used with the chemical specificity of the "naked" surface itself (for example the polystyrene that was used during synthesis of a polymer bead, or the silane surface of a glass bead), and particles that have been chemically modified to introduce more reactive surface groups, such as carboxylate (COOH) or amine (NH2) moieties. Advanced Biotechnology has a silica-based product called Magnacil used for DNA and RNA isolation using guanidine buffers. Conceptually similar to many DNA purification cartridges such as the Wizard kit from Promega Corp., this process involves binding nucleic acid to irregularly shaped magnetic silica particles of widely varying diameter, washing, and eluting. Magnacil also has been used for cleaning up polymerase chain reactions (PCR) prior to DNA sequencing. Actual photo of CPG's MPG (x 25,000) showing the porous structure of the magnetic particle. Similarly, CPG, Inc. has a 5 µm particle produced by their proprietary controlled pore glass process. These particles are totally porous borosilicate glass, with a nominal pore diameter of 50 nm. The pores in this magnetic porous glass (MPG) result in dramatically increased (almost 200 fold) surface area. To illustrate this, DYNAL's M-500 product (also a 5 µm bead) has a surface area of 7 x 10-4 m2 per 107 particles; at 60 m2 per gram, 2 µl of MPG (also approximately 107 particles) has a surface area of approximately 1.2 x 10-1 m2. Most manufacturers provide variously sized particles that have a variety of different chemical terminations; among such particles are CORTEX BIOCHEM's MagaCell (cellulose) and MagAcrolein (polyacrolein) products and CPG's silane, glyceryl, long-chain alkylamine, and hydrazide products. In general, these beads are designed as substrates for coupling high affinity, specific ligands such as streptavidin, Protein-A, or monoclonal or polyclonal antibodies. Simple adsorption of proteins is a rapid (though relatively unspecific and inefficient) method to couple a ligand to a magnetic bead. Covalent coupling is, however, preferable for many applications. A covalently bound affinity ligand is likely to be more stable, to cover the surface more completely or more evenly, and to be more efficient in terms of consumed reagents. Addition of spacers or linkers will allow biomolecules to be presented in a more flexible fashion, and careful chemistry can attach ligands in a specific orientation. There are numerous chemistries used for these couplings; many companies have published protocols and will help users with the chemistry. One unique unmodified product is a Polysciences' fluorescent bead. A polystyrene-based magnetic particle, these beads are impregnated with fluorescent label, making for easy location of the particles. The label is a proprietary yellow-green fluorophore that has fluorescein-like excitation and emission profiles. As this bead is an uncoated/non-derivatised polystyrene product, attachment of affinity ligands to the surface is via passive adsorption. CPG's MPG product has an adaptable surface. CPG's Abbie Esterman explained that controlled pore glass was originally developed for column chromatography applications. "The great thing about MPG is that the glass surface allows for almost any surface chemistry you can imagine," she said. "All of our chromatography supports, with a huge variety of particle sizes, pore dimensions, and surface properties can be made into magnetic particles as well." Silane chemistry is extremely versatile. It can be summarized by the general reaction Si-OH + R-Si-X ··> Si-O-Si-R where X is a reactive group such as alkoxy or halide and R is any functional group such as NH2, OH, or CN. As there are hundreds of R-Si-X silanes available, MPG's surface potential is almost limitless. MPG also is unusual because it is hydrophilic; it can be frozen or dried after many different surface modifications--a significant advantage over many other products for long-term storage of coupled materials. Unquestionably, the most common surface chemistry offered by magnetic bead manufacturers is COOH- or NH2- terminated polymers. Bangs Laboratories markets a variety of magnetic beads that are produced by Prolobo in France and modified in-house. Leigh Bangs believes that a variety of suppliers represented by a single specialist company, such as Bangs Laboratories, is a plus for the consumer. "We represent the best beads made--obtained from several different sources (each with its own strengths) or made by us (i.e. silica and ProActive beads)," he says. "You are not limited to only what we make. You give us your specifications and together we pick the best for your job." Bangs Laboratories has its classic magnetic microspheres and its encapsulated magnetic microspheres that are COOH- or NH2-terminated. In addition to rigorous, batch-specific QC of properties such as the titratable surface groups COOH and NH2, Bangs' classic and encapsulated magnetic microspheres are available with different magnetite contents (12 percent, 20 percent, 40 percent, and 60 percent for the classic beads and 20 percent, 30 percent, and 40 percent for the encapsulated product) allowing separation rates to be controlled in different applications. A single BioMag® Particle from PerSeptive Biosystem's, showing the patented non-uniform shape. PerSeptive Biosystems, a division of Perkin Elmer, also produces COOH- and NH2-terminated superparamagnetic beads called BioMag particles. In addition to the conventional roles in attachment of specific ligands to amine or COOH-terminated beads, BioMag was central in the development of a new molecular biology technique. Trevor Hawkins' group at the Whitehead Institute (DeAngelis et al., Nucleic Acids Research , 23 : 4742-3, 1995) noted that under specific conditions of salt and polyethylene glycol, DNA could be induced to reversibly bind to COOH-terminated magnetic beads--a process termed SPRI for solid phase reversible immobilization. The Whitehead group has used PerSeptive's BioMag beads for purification of PCR reactions and Bangs encapsulated COOH-terminated beads for the SPRI purification of M13 single stranded sequencing templates (protocols at http://www-seq.wi.mit.edu/protocols/). Advanced Biotechnology's COOH-polystyrene particles are a monodisperse bead that can also be used for purification of DNA from a variety of sources, among them blood. This COOH- terminated bead is also a good substrate for attachment of streptavidin or oligonucleotides for specific applications. A selection of ligands that bind many different biological components can be further attached to COOH- or NH2-terminated particles or to other bead surfaces. Adding a specific biological affinity ligand results in higher specificity, increased selectivity, or more stable binding of target molecules. This class of magnetic particles is simple to describe, but has thousands of varied applications. Perhaps the most generally useful are magnetic beads coated with streptavidin. The streptavidin-biotin affinity is the strongest noncovalent interaction known in nature. (A binding constant of 10-15M can almost be considered a covalent bond!). Biotin is easily incorporated into proteins and nucleic acids, giving streptavidin-coated beads wide utility. Most companies produce a streptavidin-coated particle. Other molecules with similarly general binding capabilities are Protein-A and Protein-G (which bind the Fc component of mammalian immunoglobins) and several companies also produce Protein-A or -G coated beads. Beads coated with biotin also have several obvious applications and are readily available. See Table 1 for supplier details. Magnetic particles coated with secondary antibodies, which confers increased specificity, are key to many applications. CORTEX BIOCHEM has a wide range of secondary antibodies coupled to their MagaCell and MagaBead products including anti-goat, anti-sheep, anti-chicken, anti-guinea pig, anti-human, anti-mouse, anti-rabbit, and anti-rat IgG. The hydrophilic MagaCell derivatives generally are used for protein and immunological work, while the more hydrophobic MagaBead derivatives are more commonly used to capture cells. A similar range of secondary antibodies coupled to BioMag beads is produced by PerSeptive Biosystems, and coupled to Sphero particles by Spherotech. These beads can be used to purify immunoglobins and antibody-antigen While many companies have techniques for using uncoated glass or COOH-terminated polymer beads to capture DNA and RNA, a number of products are designed specifically to isolate poly(A)+ mRNA. Most of these products feature a poly(dT) oligonucleotide coupled directly to a magnetic bead. CPG's RNA capture system, for example, is based on binding a biotinylated oligo(dT) to MPG streptavidin; while this secondary coupling might reduce efficiency, the dramatically increased surface area of the MPG overcomes this drawback. Streptavidin, Protein-A, secondary antibodies, and oligo(dT) beads all represent mechanisms that capture groups of target molecules. Many applications require capture of a single type of molecule or cell. Many products accomplish this; most of them depend on specific antibodies being coupled to the beads. It is possible to isolate cells positively (by using a bead that has an attached antibody specific to that cell) or to isolate cells by enrichment (using beads that have antibodies specific to nontarget cells). A selection of beads and antibodies produced by three companies for a variety of mammalian applications is shown in Table 2. Additionally, some companies have constructed transfection vectors that encode cell surface markers to allow isolation of transfected cells. An example of this is Miltenyi Biotec's MACSelect system that contains vectors which express cell surface antigens, such as CD4 or the mouse MHC class I surface molecule (H-2Kk). Bangs Laboratories estapor® Super Para Magnetic Microsphere Use of other specific antibodies leads to systems that can detect a wide variety of cell types. Typical of this technology is DYNAL's anti-Cryptosporidium Dynabeads, which the company claims can detect one oocyst in 10 ml water. "Our Cryptosporidium capture system is firmly established with the [Environmental Protection Agency]," said DYNAL's Heidi Israel. "We have had a product line for detection of various food-borne pathogens, such as E. coli, Salmonella, and Listeria, for several years. While other technologies, such as PCR provide heavy competition, magnetic-based purification remains a valuable capture and preenrichment approach." Clearly the future holds a plethora of rapid, sensitive, and inexpensive magnetically-based microbe, virus, and parasite detection systems. CORTEX BIOCHEM has a large number of reagents coupled to its MagaCell product that are specific to several conventional DNA purification techniques. These reagents include particles that are coated with anti-FITC, anti-digoxygenin, anti-biotin, anti-rhodamine, anti-ß galactosidase, proteinase K, RNase A and pronase E, allowing standard DNA reactions to be performed on a solid support. Similar to the oligo(dT) approach for capturing poly(A)+ mRNA, it is possible to create sequence-specific magnetic probes. The simplest method is to use CPG's approach, and synthesize a biotinylated oligonucleotide probe that can be attached to a streptavidin-coated bead. Methods to couple oligonucleotides directly to the bead surfaces also exist. While COOH and NH2 terminated beads can be thought of as ion exchange media, CORTEX BIOCHEM has a selection of products that go a step further. In addition to its MagaBead, MagAcrolein, and MagaCell products which can be used as solid-phase supports for immobilization of proteins and nucleic acids, and its MagaPhase affinity products, CORTEX has two other magnetic products that can replace conventional column chromatography in several purification techniques. MagCharc is magnetic charcoal encapsulated in a polyacrylamide matrix. MagaCharc can be used to magnetically purify small molecular weight analytes in a wide range of biological and chemical applications. Matt Pourfarzaneh, who developed this technology at CORTEX, explained that magnetic charcoal has numerous biological, chemical, and environmental applications. "We can use MagCharc to adsorb radioactive materials and dozens of environmentally important pesticides and chemicals," he said, "and after we have recovered the particles magnetically, we can desorb the captured material for analysis." The MagaPhase ion-exchange products include magnetic diethylaminoethyl (DEAE), carboxymethyl (CM), and polyethyleneimine (PEI) resins, allowing batch separation of proteins and nucleic acids. CPG's MPG particles also are available with a large number of surface features for many kinds of column chromatography, any of which can be made magnetic. CPG's Esterman explained that there are good reasons to consider using a batch-magnetic process rather than a column separation. "Small scale purifications are a typical example," she said. "Column chromatography, because of the way we have to elute the target molecules, usually results in significant dilution. With a magnetic approach, we can elute in a very small volume." In addition to eliminating column losses, keeping the purification in one tube, and being able to wash the particles thoroughly, magnetic batch separations have another important advantage over column chromatography: cell debris and other insoluble material that would rapidly foul a column is not a concern. "Our mRNA purification isolates poly(A)+ RNA directly from cell lysates," Esterman explained, "and there are not many samples that are messier than that." With a different focus, Miltenyi Biotec manufactures the smallest beads on the market--a mere 50 nm in diameter. According to Miltenyi's Woody Woodward "Miltenyi's unique magnetic "microbeads" are eight thousand to a million times smaller in volume than other magnetic beads." They use these beads in a process called MACS--magnetic cell staining. MACS beads are made with a variety of cell-specific ligands attached that can be used to label specific cell and transfected cell populations. These populations are captured using a high-gradient magnetic separation column that uses small steel wires or balls to effectively amplify an external magnetic field. Woodward explained some of MACS strengths; "Our MicroBeads are so small you do not have to remove the bead to go to culture, FACS, PCR, or a functional experiment. They will not affect cell function or viability." Spherotech also produces a selection of unusual ferromagnetic particles (COOH, goat anti-mouse IgG, and streptavidin beads). These beads are prepared using chromium dioxide rather than iron oxide. Chromium dioxide retains magnetism when exposed to a magnetic field under some conditions. These particles have found applications in some specialized areas of subcellular and receptor mechanical stress. Release of the target molecule or cell from the beads is sometimes unnecessary (for example when captured mRNA is used for solid-phase synthesis of cDNA libraries) and sometimes quite trivial (such as when DNA is eluted from a COOH-terminated bead by resuspension in a low-ionic buffer in SPRI). For many antibody-based captures, it is also often possible to release the cells from the bead by addition of an excess of the target molecule in a competitive reaction, but this results in a cell preparation that is contaminated with excess competing molecules. DYNAL has developed two systems that facilitate efficient, gentle, and complete release of cells from many of their beads. CELLection products have an antibody coupled to the Dynabeads via a nucleic acid linker. Target cells can be attached to beads using highly specific monoclonal antibodies and captured magnetically. After the captured cells have been adequately washed, they can be released from the beads using a releasing buffer that contains DNase. The beads can be removed from the preparation magnetically and the cells (which are not damaged by DNase and are completely viable) used in downstream applications. DYNAL's DETACHaBEAD technology also releases cells from the beads. Rather than employing a nucleic acid linker, it employs a competition-like mechanism of a monoclonal antibody directed against the Fab-portion of the specificity antibody that is attached to the Dynabead to disrupt the bead-antibody-target complex. As most of the products reviewed here consist of micron-sized particles, you need a relatively high magnetic field to separate them. Many of the companies mentioned in this review also supply appropriate magnetic separators. Most commonly, separators consist of plastic tube or plate holders that have embedded rare-earth (neodymium-iron-boron) magnets. Designs are available for everything from single microcentrifuge tubes, through 96-well plate holders, to complex modular designs that accommodate varying numbers, sizes, and shapes of tubes. Any company's magnetic separator should work with any magnetic particle, so make your selection based on a format that suits your application rather than based on the company that supplies your beads. (A notable exception to this is Miltenyi's MACS MicroBeads that require Miltenyi's separation columns.) However, these instruments suffer from the drawback (shared with, for example, many robotic laboratory automation systems that employ magnetic separations) that mixing to disperse the magnetic beads throughout the sample is difficult. Without efficient mixing, contact between the beads and target is limited, making binding inefficient, but too violent mixing will subject samples to excessive shear forces and is likely to damage cells and nucleic acids. Sigris Inc. (Brea, Calif.) has the only technology that overcomes these problems. Their MixSep advanced biomagnetic separation system uses a rotating magnetic field to gently disperse the magnetic particles without agitation. The medium is therefore essentially motionless, so samples are subjected to minimal mechanical disruption. MixSep also performs the incubation and magnetic separation stages. Spherotech has an instrument called the UltraMag separator system that separates and aspirates samples in 96-well microtiter plates. Their autoMag processor goes further and allows controlled incubation, separation, aspiration, and washing of samples attached to magnetic beads. Most companies, in addition to selling magnetic particles, also provide the research community with kits for specific purposes. Many of these kits, such as those for purifying DNA or RNA, simply include beads, buffers, and detailed protocols. Some go further and allow the researcher to complete a more focused and extensive operation, such as capturing RNA and performing a solid-phase cDNA synthesis or transfecting a cell line with a custom vector and purifying transfectants based on cell-surface expression of vector-determined epitopes. There are far too many kits to extensively review in an article of this length but you should be aware that they are available. All things considered, magnetic beads are becoming an integral part of today's biology lab. Regardless of your focus or of the scale of your operation, it is likely there is a magnetic bead-based protocol that can save you time and money. Bob Sinclair is a freelance writer based in Salt Lake City, Utah. He can be reached at bobsinclair@tech-write-edit.com. Table 1:Paramagnetic Particles, Table 2:Primary Antibodies

To Bead or Not To Bead: Applications of Magnetic Bead Technology

Jun 21, 1998

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