July 1, 2009 (Vol. 29, No. 13)

Gail Dutton

New Coatings, Beads, Kits, and Customized Products Expedite Research

Researchers’ needs are driving advances in the biomagnetic separations market. Recognizing that binding capacity is one of the key criteria scientists use when choosing particles, Thermo Scientific launched SeraMag® SpeedBead™ streptavidin-blocked particles this month. This bead offers many of the same qualities as the SeraMag particles, but magnetic response time occurs at least two times faster, reports Aaron Craig, marketing communications specialist.

This faster response means that clinical diagnostics assays can be performed more quickly, and that particles move more quickly through viscous solutions in molecular biology, he adds. The more rapid response time is partially attributed to the greater quantity of magnetite that is added, essentially by encapsulating the original SeraMag particle with a second layer of magnetite and a surface coating.

“These beads offer medium- to high-binding capacities, up to 55 micrograms of biotinylated IgG,” Craig says.

The greatest value of the newest addition to the SpeedBead family, however, may be the particles’ ability to reduce nonspecific binding, which yields more accurate assays.

As Craig explains, nonspecific binding of lipids, proteins, etc. can interfere with assay precision and reliability. The streptavidin-blocked particles have a new surface treatment that reduces the tendency for undesired entities bind to the bead.  Blocked beads minimize nonspecific binding in many applications while maintaining their specific biotin binding capacity.

SpeedBeads are 1 micron in diameter, and so have a slow settling rate minus the magnetic field, Craig says. These superparamagnetic beads are surfactant free and can be sonicated. They are encapsulated, so no iron is exposed.

Craig says the industry is subtly changing, as researchers gravitate toward kits. “Kits save time for our customers.” The Seradyn division of Thermo Fisher Scientific also develops custom beads. In that segment of the market, he says he is seeing interest in enzymatic coatings.

Stem Cell Separation

Invitrogen, a division of Life Technologies, launched Dynabeads® SSEA-4 last month to separate undifferentiated stem cells from differentiated cells. “SSEA-4 is a well-known marker for ESCs,” according to Paul Pickering, Ph.D., GM of Invitrogen Cell Therapy Systems. “One of the principle objectives of this is to address a need identified by the translational research community.”

In embryonic stem cell work, Dr. Pickering explains, the propensity of cells to self-replicate is “helpful in generating enough cells to be useful in clinical applications.” But, he adds, that capability can be dangerous if those cells remain among the final, differentiated cells used for therapy. Working with the Buck Institute for Age Research, scientists found these Dynabeads bind more than 99% of the undifferentiated human ESCs. Removing those cells allows researchers to work with more homogenous—and more stable—cell populations, which are better for transplantation. 

Invitrogen also recently introduced the Dynal Dynabeads® FlowComp™ Human NKp46 kit and the FlowComp™ Mouse CD49b kit, both of which use positive isolation and tube-based separation for bead-free cells. The Dynabeads® Mouse CD43 (Untouched™ B cells) kit uses negative isolation, leaving the mouse B cells in the sample, untouched by the Dynabeads.

Dynabeads® are superparamagnetic, monosized polymer beads.

Phosphopeptide Enrichment

MagPrep® beads from Merck Chimie feature magnetite content and nonporous crystal-like surfaces that let them migrate quickly in magnetic fields while binding target molecules without unspecific adsorption. To this family of products, Merck recently added the MagPrep TiO2, MagPrep P-25 Protein A, MagPrep P-25 Streptavidin, and MagPrep Carboxyl.

“MagPrep TiO2 particles are encapsulated magnetite crystals coated with a dense layer of titanium dioxide,” according to Fabrice Sultan, Ph.D., sales and marketing manager for Estapor®. With a mean diameter of 200 nm, “these particles are efficient and robust for phosphopeptide enrichment, and established protocols are available.”

“Interestingly,” Dr. Sultan continues, “different types of TiO2 materials exhibit varying enrichment profiles for phosphopeptides. MagPrep TiO2 is optimized to enrichment phospho- as well as glycopeptides before downstream analysis. The particles are highly selective for monophosphorylated and multiphosphorylated peptides, and show low affinity toward acidic peptides. The MALDI mass spectra demonstrate the highly selective enrichment of phosphopeptides analyzing a tryptic digest with 1:30 molar ratio of phosphoprotein to nonphosphoprotein and detecting phosphopeptides in the 0.01 pmol range. Thus, TiO2 magnetic particles can be used for automated protein phosphorylation studies on complex biological samples.”

MagPrep P-25 Protein A is a suspension of 25 nm particles with protein A covalently coupled on their surface, to capture immunoglobulin from a variety of samples and can be used for immunoprecipitation of target antigens. “MagPrep P-25 Streptavidin can be used for affinity capture of biotinylated molecules such as proteins and nucleic acids labeled with biotin,” Dr. Sultan says. MagPrep P-25 Carboxyl can be used for covalent attachment of ligands to the surface. “The amount of protein or IgG that can be immobilized on the surface is in the range of 100 µg/mg particles,” Dr. Sultan says. Immobilization protocols for this product are available upon request, he adds.

Virus Capture

Ademtech offers Viro-Adembeads, Bio-Adembeads PAG-Immunoprecipitation, and two new streptavidin surfaces—recombinant streptavidin and streptavidin.

Viro-Adembeads capture viruses in only 30 minutes, according to the company. Because the separation method is gentle, live infectious viruses can be isolated and cultured on the bead, increasing and accelerating virus production, it reports.

In Ademtech’s tests involving HIV-1 production spiked human plasma, the culture produced using the Viro-Adembeads yielded 465,050 copies of the virus at day 11, versus only 14,059 copies using virus obtain by centrifugation, and only 343 in the control sample. Reproducibility was slightly higher using Viro-Adembeads, the company says. Sensitivity was shown as 1×105 with centrifugation, compared to 1×108 in patients with an initial viral load of 106.

Bio-Adembeads PAG combines the IgG binding domains of both protein A and protein G into one genetically engineered particle for immunoprecipitation. Protein AG binds to all IgG subclasses from multiple mammalian species, but lacks albumin, cell-wall binding regions, and other non-specific binding sites. Consequently, specificity is high for polyclonal and monoclonal IgG antibodies, and background noise is minimal. This gentle separation system doesn’t require precleaning steps or centrifugation, and reduces washing steps and incubation time.

“The challenge is to propose the widest choice of magnetic nanoparticles to comply with classical immunoassay requirements and with new generations of diagnostics device requirements,” according to Sandrine Godichaud, product manager. “Ademtech produces uniform, mono-sized and spherical particles with highly defined and consistent product characteristics,” she says, “that ensure a high level of reproducibility and predictable results.” The company offers magnetic nanoparticles that include 100, 200, 300, and 500 nm sizes with surface coatings that include COOH, NH2, streptavidin, and silica.

Adembeads are uniform nanoparticles presenting a magnetic iron (>70%) and a polymer shell, reports Ademtech.

New Coatings

Bangs Labs/Polysciences has introduced a new type of magnetic particle and new coatings for existing biomagnetic separation products this year, says Kathy Turner, technical services manager. These additions enhance users’ ability to select the best particle for their application, she adds, “whether it’s a magnetic particle assay, cell separation, or protein isolation.”

“Our newest magnetic particle is the ProMag,” Turner says. A 1 micron version will launch this summer, and the 3 micron version is already available. “These spherical beads are highly uniform in diameter and have a surface topography that yields a high surface area and specific binding, as well as low nonspecific binding without the need for surfactants.” One milliliter volumes can be completely separated from a fully dispersed state within 15 seconds. A range of magnetic separators and separation systems allow protocols optimization for small- or large-scale applications.

Coatings for the BioMag® Plus particle line now include wheat germ agglutinin (WGA) and concanavalin A (Con A), in addition to Bang’s primary and secondary antibody coatings. “These ligands may be used to isolate cells and biomolecules that contain specific classes of sugars,” Turner says.

“Common uses for WGA and ConA BioMag include isolating glycosylated proteins from serum or cell lysate, and studying other lectin/glycan-mediated processes.” The BioMag particles approximate 1.5 micron diameters and irregular morphology produces a surface area of more than 100 m2/g.

Turner adds that Bangs Labs is seeing particle coatings being used in emerging fields. “For example, immunoprecipitation is a well-established technique that has long been accomplished with protein A or G matrices. Within the field of proteomics, we’re seeing investigators use traditional microparticles and protocols to support this developing area.”

Dexter Magnetic Technologies is working to develop scalable magnetic separation processes using fixed magnets. “Customers want constant packing density in all configurations. Therefore, we have to ensure the product is scalable,” notes Doug Hartl, business manager, medical products.

The work to develop scalable magnetic separation processes, so far, is being performed as custom-development projects. Vessel size currently may scale from about a half liter to one liter, with repeatable performance and outcomes within that range. “It’s fairly early on in the development process,” Hartl says. If interest in scalable biomagnetic separations continues to grow, he speculates that Dexter may consider adding a scalable separator to its standard line of separators.

At Dexter, LifeSept is the standard line for biomagnetic separations involving the biotech industry. The separators accept particles as small as 0.8 microns.

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