July 1, 2008 (Vol. 28, No. 13)

Manufacturers Are Rendering This Versatile Technology More Widely Available

Biomagnetic separations are expanding into nontraditional fields including neurology and proteomics as new disciplines discover the availability of gentle ways to separate cells and even classes of cells. It seems that many people who have never used magnetic separation are now considering this technology. To further boost utility, manufacturers are developing new ways to maximize biomagnetic separations’ potential.

As Sandrine Godichaud, product manager, Ademtech (www.ademtech.com), points out, “Magnetic particles are a powerful and versatile diagnostic tool and are particularly indicated for use in a number of clinical research applications such as microbiology, immunology, proteomics, molecular biology, drug discovery, and cancer research.

“Magnetic nanoparticles provide interesting perspectives for realizing novel diagnostic sensors because they allow the detection of magnetic particles specifically bound to the biological target at low concentration. The nature of the magnetic nanoparticles plays a crucial role in developing a sensitive and specific magnetic sensor.” With such benefits, it’s no wonder companies are expanding their uses.

Automation is entering the field of biomagnetic particle separations. “A lot of people have requirements for sample prep upfront, and biomagnetic separations are quite easy to automate,” notes Rick Galloway, director of particle technology at Seradyn (www.thermo.com/seradyn), a subsidiary of Thermo Fisher. Likewise, “new application fields are exploding,” he says.

Proteomics is an intense area of research as scientists drill deeper and deeper to identify proteins or groups of proteins that indicate a disease state or disease predisposition. “Some of these proteins are present in extremely small quantities,” Galloway notes. So, considering that human blood contains hundreds of thousands of proteins, mass spectrometry—the traditional quantification method—is impractical. Instead, by applying biomagnetic separation upstream, mass spec analysis is improved by allowing it to focus on the proteins of interest.”

Nonspecific binding is a universal problem for biomagnetic separations, Galloway says, and Thermo Fisher has next-stage products in development to minimize nonspecific binding. Thermo’s strategy involves the particle itself.

Rather than making a coated particle that binds to a specific target and then adding a compound to block nonspecific binding, we’re doing the reverse—making a blocked particle and then making it target specific,” he remarks.

ThermoFisher is also developing surface coatings specific to various classes of targets rather than to specific targets themselves. For example, Galloway says, the particle coating may bind all phosphopeptides in the solution, which would benefit sample prep for mass spec.

Fluorescent Beads

The need for compromise between affinity and specificity is also diminishing. “The technology has advanced far enough so that magnetic beads must perform under a broad range of conditions,” notes Chad Owen, vp, Bangs Laboratories (www.bangslabs.com), which is owned by Polysciences (www.polysciences.com). So, for example, the same streptavidin-coated bead would perform well for virtually any assay appropriate to this coating, he says.

At Bangs, Owen reports a strong and growing interest in using biomagnetic microspheres. Using fluorescent magnetic beads for flow cytometry applications fulfills requirements related to fluorescence while permitting easy, efficient magnetic separations. “Not all researchers know they’re available,” Owen says, so creating awareness is a challenge. That may change now that the fluorescent beads are available on a commercial scale.

Another area gaining attention is proteomics, in which microbeads can replace traditional column-based separation. Polysciences’ high-capacity WGA (wheat germ agglutinin) kit, based on its BioMag® bead, is an item of interest for removing glycan-presenting cells and glycoproteins.

There’s also continual growth in diagnostics, using Polysciences’ProMag™ line, Owen says. To support this, Polysciences now has a 1 micron ProMag bead in stability testing. It provides more surface area per weight than the company’s popular 3 micron ProMag beads, making it advantageous in nucleic acid-based diagnostics and separations, Owens explains. “The challenge,” he says, “is that smaller beads require a higher percentage of magnetite than larger beads, so their density increases.” Bangs is looking at various forms of magnetic material to achieve the optimum balance between density and magnetic properties.

Raising Awareness

Miltenyi Biotec(www.miltenyibiotec.com) is expanding its core product offerings for immunologists. In fact, multiple kits have been released virtually every month for the past year. “We’re a science-driven organization,” points out Kirt Braun, Ph.D., marketing manager. “As scientists tease apart the mechanics of the immune system, they come across cell subtypes of interest,” for which biomagnetic separation makes sense, particularly when viewed against traditional flow cytometry, which can be cumbersome and may have a negative impact on individual cells.

Biomagnetic separations and their streamlined workflow, however, are new to many researchers outside the world of immunology. “Awareness is the big challenge,” Dr. Braun says. Researchers often are astounded that biomagnetic separations can be used in their own particular fields.

“Therefore,” Dr. Braun reports, “Miltenyi is extending its reach to new markets. Neural cell isolation, for instance, has undergone a revolution. Researchers are interested in isolating specific cell populations within the brain much faster and more reliably than with selection by culture. So, we offer a complete workflow solution in the form of kits to dissociate the neural tissues and cells into viable, single-cell suspensions.”

Miltenyi released kits utilizing trypsin or papain, plus the automated gentleMACS™ Dissociator for mouse, rat, and human tissue. The technology can also be used for spleen and liver tissue, with protocols for lung and tumor tissue on the horizon. Miltenyi has also a growing portfolio of MicroBeads to isolate neural cell subsets, such as anti-PSA-NCAM MicroBeads to isolate neuronal restricted precursors and anti-A2B5 MicroBeads and antibodies for the positive selection and analysis of glial-restricted precursor cells.

The focus in stem cell research is continuing, as the demand for supplies to support it is growing at 10% or more per year, according to Dr. Braun. Miltenyi recently released anti-Prominin-1 MicroBeads and antibodies to isolate and analyze mouse neural stem and progenitor cells and the CD146 MicroBead Kit to isolate human mesenchymal stem cells from lipoaspirates and other tissue sources.

“An increasing numbers of scientists are focusing on the T-cell regulatory pathways, as this pathway is of great importance to better understanding and modulating autoimmune disease,” Dr. Braun continues. Miltenyi’s portfolio of products for the study of T cells and T regs now includes a kit for the isolation of a new T-cell subset, characterized as CD4+CD25+ and CD127dim, which helps researchers focus their efforts and drill down through the immune system to better understand its regulation and thus certain disease mechanisms.

The MACS® Cytokine Secretion Assay (CSA) technology will also continue to expand to encompass a greater variety of the cells involved in regulating the immune system. “Miltenyi’s CSA products isolate antigen-specific cells. In the next few months, new CSA kits will be released that will enable researchers to isolate antigen-specific cells based on the secretion of interferon alpha as well as interleukin 17,” Dr. Braun adds.

At Invitrogen Dynal (www.invitrogen.com/dynal), applications for Dynabeads Protein A and Dynabeads® Protein G have expanded into immunoprecipitation. “As a response to the increased interest in immunoprecipitation (IP), co-IP, chip and other pull-downs, we will be launching dedicated kits for immunoprecipitation this fall,” according to Haege Wetterhus, business segment director, molecular separations.

“Another hot topic is sample preparation for peptide profiling. Processes for sample collection, preparation, and analysis are being standardized in biomarker discovery and in clinical proteomics. The Dynabeads for magnetic bead-based chromatography were launched to meet the need for pre-massSpec sample prep. We will be expanding this product range even further in the future,” Wetterhus says.

Invitrogen Dynal is seeing a trend to use primary cells to create studies that more closely resemble in vivo studies. The aim is to remove outside influences. Positive cell isolation allows direct pullout—a benefit of the FlowComp™ product line—thus removing the risk of contamination with iron, dextran, or other potentially harmful structures. Because positive cell isolation has minimal impact on cells, they maintain functional characteristics and are suitable for all downstream applications,” notes Øystein Aamellem, Ph.D., business segment director, cell separation and expansion.

The FlowComp product line was launched last summer with human and mouse CD4 and CD8 specific cell isolation. The product line has since been extended with Dynabeads FlowComp Flexi and Dynabeads FlowComp Human NKp46, Aamellem says. Two more FlowComp products were slated for launch last month—Dynabeads FlowComp Mouse CD49b (+mouse NK cells) and Dynabeads FlowComp Mouse CD4+CD25+ T reg Cells (+mouse regulatory T cells).

The Dynabeads negative isolation kits are being rebranded as the Dynabeads Untouched™ line and are being reformatted to double the isolation capacity. The reformatted human CD4 kit launched in March with greater than 90% purity, 70% average recovery, and about 98% viability, Aamellem reports. The CD8 kit launched at the same time and the human T-cells kit launched last month.

“Dynabeads T-cell expansion products allow activation and/or expansion of naive and memory T cells, T-cell clones, regulatory T cells, NKT cells, and CTLs from mouse and human samples,” Aamellem adds. Originally developed for immunotherapeutics, they are being used in the clinical trial arena and in the clinic to standardize blood cell assays. Blood cell separation has been a major barrier in improving high-throughput screening and developing standardized blood cell assays and other clinical diagnostics.

Ademtech is optimizing the key parameters of beads for biosensor applications. For example, if the magnetic core’s iron content exceeds 70%, detection is enhanced. A homogenous, smooth surface reduces nonspecific binding and the uniform, nanometric bead size decreases particle sedimentation rates while increasing specific surface area.

These magnetic nanoparticles range in size from 100 to 500 nm, with surfaces that include COOH, NH2, streptavidin, and silica “to comply with biosensors development requirements,” Godichaud explains. “We have developed magnetic beads that have a specific surface polymer designed for DNA capture by electrostatic interactions and compatible with direct PCR and real-time PCR without the need to perform an elution step.”

Recent additions to Ademtech’s product family include Bio-Adembeads Streptavidin plus two levels of biotin-binding capacities for 200 and 300 nm particles, and a new streptavidin 100 nm particle.

Need Drives Automation

Dexter Magnetic Technologies (www.dextermag.com) has focused on separation equipment, betting that “future growth is in automated particle separation equipment,” according to Doug Hartl, business manager for medical and LifeSep products. The attention to automation is driven by the “need for high-throughput analysis, the demand for new drug development, and the demand for higher testing volumes in the diagnostic arena,” he says.

Much of Dexter’s work, Hartl states, is with diagnostic equipment manufacturers, providing custom-engineered design assistance on such aspects as sophisticated magnetic particle separation subsystems. Some requests, for example, are for systems that perform separations at specific rates and for systems in which the magnetic beads are placed at specific positions in a tube or vessel. Depending upon the machine, he explains, flow-through separation may be beneficial, or the client may prefer particles to be placed on a specific position within a vessel.

“Customers are looking for ways to manipulate magnetic particles to advance innovation,” he adds. Ongoing improvements in magnetic properties make miniaturization possible, so researchers may achieve more efficient results using less magnetic material.

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