July 1, 2005 (Vol. 25, No. 13)

Gail Dutton

Nano-sized Beads and New Coatings Increase Sensitivity and Facilitate Analysis and Automation

Merck Chimie (www.merckchimie.fr) introduced its new 1.2 to 1.5 m monodispersed superparamagnetic beads in June at its third international meeting in San Diego. According to Fabrice Sultan, Ph.D., sales and marketing manager, “Their COOH-modified surface is stable and provides an active surface to which immunoglobulin, proteins, or other ligands can bind easily and strongly.

“With a coefficient of variation less than 7%, this new product will allow the development of more reproducible and more accurate immunoassays.” These new, smaller beads retain the same surface area and same magnetic properties as previous generations of Merck Chimie’s beads.

Nano-sized beads are among this year’s trends in biomagnetic particles. Coupled with new or improved coatings, the beads being introduced today offer not only greater sensitivities but open the doors to more effective downstream analysis and automation.

A new, hydrophobic superparamagnetic bead recently introduced by Merck Chimie can be considered preactivated, and offers “very strong binding,” Dr. Sultan says, when coupled with immunoglobulin or proteins. When they were coated with antibodies or streptavidin, “preliminary results showed very good data,” he says.

“With such beads, immunoassay developers could reduce the risk of antibody or protein leakage from the beads during storage, thus increasing the reagent shelf life.” The 0.7 to 1.3 m beads have a ferrite content of 3050%, making them amenable to automated immunoassays.

Merck Chimie also introduced nanoparticle-sized beads in 150-nm, 200-nm and 300-nm sizes. “They offer a great surface area, a low sedimentation rate, high ferrite content, and a good magnetic signature, and also increase the sensitivity of the assays,” Dr. Sultan says. Applications include cell sorting, immunoprecipitation, quantitative lateral flow assays, and biosensors.

Biomarker Discovery

Bruker Daltonics (Billerica, MA) introduced the next level of the company’s ClinProt solution for advanced biomarker discovery, identification, and validation in clinical proteomics at last month’s “American Society for Mass Spectrometry” (ASMS) meeting, held in San Antonio.

The firm’s new ClinProt Large-Protein Beads are designed to overcome previous limitations of biomarker discovery to peptide and small proteins and to exploit the full mass range of up to several 100 kDaltons supported by Bruker Daltonics’ MALDI-TOF mass spectrometers.

New ClinProt Glyco-Beads bridge the gap between clinical proteomics and glycomics, the company claims. The Glyco-Beads offer multiple methods for glyco-protein enrichment, including both chemical- or lectin-capture strategies.

The new Antibody-Capture Beads allow the specific enrichment of different protein groups with the same antigen specificity or even of specific proteins, thus giving access to low-abundance proteins in plasma.

Magnetic Bead Chromatography

Dynal Biotech (www.dynalbiotech.com), now a subsidiary of Invitrogen, is introducing several key products this year. “Magnetic bead chromatography is one of the new developments here,” notes Lars Korsnes, Ph.D., business area manager, in vitro diagnostics. This new product line isolates proteins and large peptides using affinity interactions, thus eliminating the need for columns or centrifuges and, Dr. Korsnes says, works with a few microliters of samples.

The first two products within this familywith more plannedinclude the Dynabeads RPC (reversed-phase chromatography) Protein for serum profiling and Dynabeads WCX (weak cation exchange).

The RPC products are designed for serum profiling and are particularly effective for top-down proteomics strategies. The WCX beads separate proteins by exploiting differences in net charge. Key benefits include concentrating and fractionating the protein samples, desalting and removing contaminants, and preparing the sample for mass spectrometry analysis.

A poster presented at the ASMS noted that, using the RPC Protein and WCX beads, “even nonautomated protocol sample preparation is reproducible,” and “both high and low abundant protein peaks are detected, which are above general instrument noise.”

In fractionating a cell lysate from a human cancer cell line, analyzed by 2-D gel electrophoresis, 2,211 spots were detected, compared to only 655 spots from an unfractionated lysate. Because the beads generate different profiles, they may be used in parallel on the same serum samples to increase the chance of identifying novel biomarkers.

This summer, Dynal is also introducing the Dynabeads Tcap antigen-specific CD8+ T cells isolation kit. These beads, coated with peptide-loaded recombinant HLA-A2 molecules, isolate antigen-specific CD8+ cells with high yield, which then can be expanded.

They are available for Epstein Barr virus, cytomegalovirus, and a range of cancer antigens. The cells can be isolated directly from mononuclear cells or, alternatively, samples with low cell concentrations can be depleted of unwanted cells before isolation and expansion.

Dynal also recently introduced ready-to-use artificial antigen-presenting cells to re-stimulate human and mouse T cells. Dynabeads provide consistent, simultaneous signals to TCR/CD3 and CD28 for full activation and expansion of human or mouse T cells in culture, eliminating negative signaling and feeder cell contamination and maintenance.

Anti-Legionella beads, another new Dynal product, are designed for selective separation and concentration of Legionella species for immunomagnetic separation. A viable yield can be obtained from inocula containing less than 100 colony-forming units per milliliter of test sample in clean water.

Seradyn (www.seradyn.com) considers its Sera-Mag magnetic bead “a workhorse,” according to Rick Galloway, director of particle technology. “It offers high-binding capacity, chemical stability, and long-term shelf life.”

New ligand coatings are being developed for this bead by Seradyn’s still-confidential partners to enhance specificity, efficiency, and purity in the areas of endotoxin removal, virus concentration, and protein isolation. The new products are likely to be released by Seradyn in late 2005 or early 2006, Galloway says. As yet, the details are being closely held.

That said, the emerging endotoxin removal product is particularly beneficial in transfection applications, where researchers are trying to isolate specific nucleic acid sequences. Endotoxins can ruin the transfection process, so they must be removed. Endotoxin removal can be accomplished by HPLC or other laborious techniques and may take days. Or, Galloway says, it can be accomplished in a few hours using biomagnetic separations.

“One of the challenges in viral load testing involves sensitivity versus sample size,” he says. “When trying to determine the number of viral copies per milliliter of sample, you can’t pour 10 mL into the reaction, so you need to concentrate the virus copies,” into an appropriately sized sample. Seradyn’s Sera-Mag magnetic beads have enabled its partners to develop a virus concentration product that is expected to resolve that issue.

For protein isolation, new tags are being developed to augment histidine and GST. Assuring “purity and specificity are key,” Galloway says.

mRNA Purification

Ademtech (www.ademtech. com) recently introduced the mRNAdembeads Purification kit for mRNA purification starting from total RNA. “The kit isolates the messengers in 20 minutes, and the RT-PCR can be directly performed in the presence of nanoparticles,” according to Jerome Jolly, product manager. It is fully compatible with amplification enzymes activity.

“High-purity mRNA can be isolated from as little as 1 g of total RNA and concentrated in small volume less than 15 L,” he says. Consequently, low expressed mRNA, such as the DFTR gene from pulmonary cells, can be purified, which is useful for sensitive microarrays.

“Soon,” Jolly says, “Ademtech will present another version of this kit to directly purify mRNA from cell lysate and blood, reducing the amount of starting material to 100 cells.” By year’s end, the technology will be adapted for standard liquid-handling robots. Ademtech has also developed a new magnet, the Adem-Mag MSV, to facilitate purification of a large number of samples simultaneously.

Ademtech’s superparamagnetic nanoparticles are available in sizes of 100, 200, 300, and 500 nm, all with a coefficient of variation of less than 20% and a magnetic oxide content greater than 70%. Surface area is up to 15 m2/g, for high sensitivity.

“This offers an easy, automatable, sensitive tool for immunoassays without sedimentation or aggregation associated with larger and polydispersed magnetic particles,” Jolly says.

“The technology has been applied to produce different surface chemistriesCOOH or NH2or with biological activities brought by streptavidin, antibodies, or protein AG dedicated to proteomics and cell sorting.”

At Bangs Laboratories (www. bangslabs.com), the new QuantumPlex M superparamagnetic multiplexing bead array is based on a polymer-based 6-micron magnetic particle dyed with the company’s proprietary fluorophore, Starfire Red.

Designed for the flow cytometric analysis of a sample for multiple analytes or high throughput screening of multiple samples, the QuantumPlex M platform lets users see five distinct fluorescence peaks. Researchers can decorate particles with antibodies or DNA probes, for example, and excite those particles using 488- or 633-nm lasers.

Small labs, in particular, will benefit, according to Kathy Turner, technical services, because there is no additional equipment to buy. The particles work with any standard flow cytometer.

The BioMag ProMax product family recently added the Albumin Removal Kit and the Serum IgG Removal Kit. Albumin accounts for 5070% of the total protein in serum, and IgG accounts for 1020%. Removing these proteins facilitates detection of less abundant proteins in serum. The kits require no sample pretreatment, no columns, and no centrifugation.

Bangs also is continuing to expand its BioMag product line, Turner says. The particles are more than 90% iron oxide, irregularly shaped for a “tremendous” surface area, silanized to enhance functionality and bonding, and about 1.5 microns in diameter. Other products are in development but are, as yet, proprietary.

WaveSense (www.wavesense. com) focuses on xenographic retention chromatography, which fuses multiple dissimilar separations technologiesnotably magnetic separation, lateral flow separation immunoaffinity, and nucleoaffinity. “Both cellular and molecular assays can be performed with the simplicity of a test strip,” according to Christopher Feistel, CEO. “Performance surpasses microtitre and flow cytometry formats.”

The system, Feistel says, is akin to “a test strip on a rail.” It creates flow resistance at the nano-scale and “works with the applied magnetic field to arrest magnetic particles coated with antibodies or oligo probes specific for the target cell or molecule,” pulling the target into alignment for analysis, effectively concentrating the signal.

Only magnetic particles are captured in the flow path, he emphasizes, resulting in “low target loss.” By capturing the beads in a lateral flow path, there is no cellular manipulation or washing away of nontarget cells. Sensitivity is as great as one in 1 billion cells, versus one in 10,000 cells for flow cytometry.

Throughout is also high1 mL (about 4 billion cells) can be processed in 30 seconds, compared to two or more hours for flow cytometry, he says. The recovery rate is as high as 90% of the target cells.

Therefore, Feistel says, “We can use readily available fluid-handling systems, and the technology can then become a high throughput cell sorter with sensitive detection devices for specimen volumes ranging from nanoliters to several milliliters. This is ideal for high content cellular assays.”

X-ray chromatography research (XCR) kits to detect tumor cells in blood and bone marrow have just been released for drug discovery. XRC tool kits will be released for researchers later this year, allowing them greater flexibility.

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