July 1, 2007 (Vol. 27, No. 13)
Faster, Cheaper, and More Efficient Identification Will Streamline Discovery
Protein biomarkers remains appealing to drug developers because of the promise of early elimination of ineffective or toxic compounds, improved target validation, and creation of cellular assays for drug discovery. However, the difficulty and costs of characterizing and validating new biomarkers has held back rapid development in this field. Now, new biomagnetic beads are providing ways to identify biomarkers faster, more efficiently, and cheaper.
“For biomarker discovery, investigators can coat beads with the ligand of interest, and either screen samples to identify binders to that ligand, or use coated beads for purifying target.” She adds that in the case of lectin-coated beads, like the company’s soon-to-be-launched BioMag® WGA (wheat germ aglutenin), the lectin binds to its carbohydrate-binding partner. “This is useful for purification of glycoproteins from cell lysate for downstream applications, as well as for certain cell separations that would be more of a biomarker-type application.”
The company offers a suite of various beads. Its BioMag Superparamagnetic particles are used for separation of cells, organelles, proteins, nucleic acids, etc. These irregularly shaped iron-oxide particles contain more than 90% iron oxide for faster magnetic separation. They are covered with a coating that provides functional groups for attachment of proteins or antibodies. BioMag binding proteins include: biotin, Protein A, Protein G, streptavadin, and nuclease-free streptavadin.
“We focus on high coating ability, low, nonspecific binding, and fast pulling speeds so they respond well to the magnet,” states Turner. “It’s up to the customer to choose suitable coatings and optimize them.” Advantages of the beads include a high surface area and an easy format to work with. They also provide great flexibility in that the user can take a population and coat it with different molecules, store it as a wet reagent for significant periods of time, and use it for separation assays.
“A big advantage with microspheres versus a microtiter format is the tremendous surface area you’re presenting to the sample. So there are high capture efficiencies and good binding dynamics.” Also, since most applications involve an automated magnetic separation system, there is a major savings in labor and time, says Turner.
Many customers require magnetic beads with increased speed of response to a magnet, thus Thermo Scientific Seradyn’s (www.seradyn.com) newest member of the Sera-Mag® product line is SpeedBeads, which the company reports is twice as fast as the original particles. These 1-micron carboxylate-modified base particles are made by a core-shell process. “People like the beads to be the same size so they move at the same speed,” explains Rick Galloway, director, particle technology. High-binding capacity is another characteristic customers expect. In order to increase surface area, the beads have a bumpy, cauliflower-like surface.
“All these valleys add greater surface area without changing the diameter. One way others have attempted to increase surface area is to make smaller diameter particles. But the smaller particles respond more slowly to a magnet and that is not good. The way we build our particles is such that the lumpy surface makes them unique, yet they retain the one-micron speed—a nice balance between speed and response,” adds Galloway.
The settling rate in the absence of a magnetic field is yet another important characteristic. SpeedBeads have a slow settling rate—the particles should be floating around and reacting with the target, not dropping to the bottom of the container, explains Galloway.
The most common configurations customers are using for biomarker discovery are SpeedBeads with streptavidin and specific antibodies that have been biotinylated. These feature covalently bound streptavidin with four levels of biotin binding capacity and a shelf life up to five years. The carboxylate-modified beads are the company’s base bead; many customers add their own coating to the surface. “The real optimization lies in tweaking the coating concentration. We’re doing more custom coupling for companies.” The next area of focus for the company will be different surface coatings—more functional, active compounds.
Chromatin Immunoprecipitation Kit
Ademtech (www.ademtech.com) introduced a new kit, ChIP-Adem-Kit (chromatin immunoprecipitation), specifically designed to decrease nonspecific binding and increase signal-to-noise ratio. “This is important because when you have immunoprecipitation you can have non-specificity that can come from DNA and/or protein,” says Sandrine Godichaud, product manager. The kit includes magnetic beads coated with Protein A and Protein G, to bind antibody in order to recognize the target protein, as well as buffers and reagents required to perform the immunoprecipitation step.
“Protein/DNA complexes are fixed by cross-linking with formaldehyde and the chromatin is sheared into DNA fragments. DNA and protein complexes are then immunoprecipitated using an antibody specific of the targeted protein. DNA is purified from the isolated chromatin and specific genomic regions are detected by PCR or real-time PCR,” Godichaud explains.
The beads are monodispersed, superparamagnetic, core-shell beads in the range of 100–500 nm and are homogenous with a smooth surface to reduce nonspecific binding. The small size reduces particle sedimentation rate and increases specific surface area.
ChIP is a tool to detect association of individual proteins with specific genomic regions in vivo. According to Ademtech, it saves time and cost by eliminating chromatin precleaning, replacing the spin steps with fast magnetic separations, and allowing for simultaneous multiple ChIP assays, which facilitates simultaneous monitoring of many genomic events.
Applications include combination with microarray technology to identify the location of specific proteins on a genome-wide basis, ascertaining how regulatory proteins control gene activities, studying the association of proteins with specific genomic regions, and analyzing protein pathways to aid in the discovery of biomarkers and drug development.
In addition to offering a wide range of magnetic beads at various sizes, surfaces, ferrite content, stability, batch capacity, as well as custom designs, Merck Chimie (www.merck.fr) has introduced three new microspheres. “We offer the largest range in terms of sizes,” says Fabrice Sultan, Ph.D., international sales and marketing manager. These range from 100 nm (cell separation, biochips) to 2–3 µm for separation of products with a high weight. Various surfaces include COOH, NH2, Epoxy, Tosyl, and SH groups. “This offers our customers the ability to optimize their reagent in terms of binding capacity, signal-to-noise ratio, sensitivity, immuno-reactivity, and cost,” explains Dr. Sultan.
The hydrophobic magnetic beads are well-suited for biomarker discovery. “Proteins with a molecular weight of greater than 40 Kda offer many hydrophobic areas and easily bind to our beads. We also have beads coated with anti-IgG, which are useful for immunoprecipitation.” Overall, the advantage beads provide is the ability to work in 3-D, increasing the surface and sensitivity, along with high reaction kinetics of the assay.
Bioestapor Rec-Streptavidin 1.0-µm beads offer high binding capacity and are nuclease- and protease-free—essential for nucleic acid and proteomics applications. A high iron content (greater than 40%) allows rapid collection from the aqueous phase and makes the beads compatible with either manual or automated platforms.
The small COOH-modified Super-Paramagnetic Estapor® Microspheres are 160 nm and offer great surface area, low sedimentation rate, high ferrite content, and great magnetic signature, according to the company. These are used in cell sorting, immunoprecipitation, and quantitative lateral flow assays. Hydrophobic superparamagnetic beads, the last of the new products, allow strong, direct binding to immunoglobulin or proteins. The company says that with these beads, immunoassay developers can reduce the risk of antibody or protein leakage during storage.
Magnetic Bead Chromatography
A new line of magnetic bead chromatography products was recently introduced by Invitrogen Dynal (www.invitrogen.com/dynal). “We have discovered that the biomarker community has problems creating reproducible platforms, and ones that are low in cost and time,” states Beate Johnsen, senior product manager, molecular separations. The Dynal® Peptide Profiler is a high-throughput serum-profiling package using the Tecan®robot platform with Dynabeads® RPC 18. This captures peptides directly from serum and/or plasma for mass spectrometry and generates disease-specific peptide profiles for biomarker discovery. It can process 96 samples in parallel within 27 hours, according to the company.
“Data has shown that you can actually compare biomarkers from different dates with these beads because there is low variation and high reproducibility,” Johnsen adds. “The protocol is simple because you just add serum or any type of biological fluid to the bead and use the magnets to pull out the beads and then use the sample directly in mass spectrometry.”
Dynabeads RPC 18 are strong hydrophobic reversed-phase, uniform monodisbursed 1-µm magnetic beads for preparation of proteins between 700 and 12,000 Daltons. “Many customers complain that other beads have lot-to-lot variation because they don’t have monodisbursed beads,” Johnsen explains.
Several other recently introduced magnetic beads for the preparation of complex protein and peptide mixtures include Dynabeads WCX (weak cation exchange), Dynabeads PRC protein (weak hydrophobic reversed-phase bead for preparing samples of large peptides greater than 5,000 Da), and the Dynabeads SAX—a strong anion exchange magnetic bead.
“What we don’t have is a method of doing different types of protein fractionation. Most people are talking about being able to fractionate the proteins so you will get a clear picture of the low-expression proteins. If you have a range of products to try, maybe a certain bead will show the biomarker pattern you are looking for,” Johnsen summarizes.
Miltenyi Biotech (www.miltenyibiotec.com) MACS® technology (magnetically activated cell separation) was developed in 1989. MACS Microbeads are superparamagnetic particles, 50 nanometers in size (about the size of a small virus). Sample cells are magnetically labeled with the beads and a magnetic field is induced on the steel matrix of the MACS column when it is placed in the MACS Separator. While the magnetically labeled cells are retained in the column, the unlabeled cells run through, the column is removed, and the retained microbead-labeled cells can be eluted.
“Microbeads offer several advantages,” explains Tim Stolle, Ph.D., product manager, research marketing. “Their small size makes the MACS technology compatible with flow cytometric analysis; the beads do not alter the structure, function, or activity status of the labeled cells; and since they are composed of a biodegradable matrix, it is not necessary to remove the beads from the cells after the separation process.” Isolated cells can be directly used for cell culture and in vivo experiments.
µMACS™ Microbeads perform analytical protein isolation in less than two hours. To confirm protein-protein interactions, the company offers the µMACS Tag Isolation Kits to detect epitope-tagged proteins. The beads are coupled to specific antibodies to capture His-, HA-, c-myc-, GRP-, or GST-tagged proteins. The µMACS Streptavidin Kit performs magnetic separation of any interacting molecule with a biotinylated probe. The µMACS Protein A and Protein G microbeads allow analytical isolation of pure target proteins with almost any antibody. These kits are used with the µColumn designed for small-scale molecular biology and the µMACs Separator, which holds up to four µColumns.