by Caitlin Smith
Separating proteins or nucleic acids is reliably accomplished by gel electrophoresis – so why is this trusty method in danger of being supplanted by a high-tech cousin? Profiling assays for analyzing mixtures of proteins or nucleic acids, based on binding to tags on tiny beads, are quickly becoming invaluable for profiling many species simultaneously using multiplexing technology. The ability to separate more species, with greater accuracy and speed than with conventional methods, can result in very attractive changes to the workflow of a lab.
Despite such advantages, bead-based profiling assays are not increasing in popularity as fast as one might expect. “I think the biggest challenge for these assays is hesitation among researchers to accept this new technology,” says Mike Vespremi, product manager at Assay Designs. “But with developments in conjugation chemistries and differing bead technologies, I believe it is starting to gain more widespread acceptance in the research community.” Indeed, the degree of multiplexing that this field is achieving would prompt any researcher to take a second look.
Sample preparation and size
All assays, of course, begin with a quality starting sample. According to Fiona Coats, VP of business development at Marligen Biosciences, sample preparation plays an important role in the limitations of the assay itself. “As with all platforms, the challenge comes from making sure that the assays are as sensitive as possible,” she says. “These limitations usually do not come from the platform, but from sample preparation. The industry has continued to see better, more efficient sample preparation and amplification technologies.”
Maria Wasienko, product manager at Millipore, agrees that sample preparation is an important step in the protocol. “One of the biggest challenges for bead-based assays is eliminating variability with careful upstream sample processing,” she says. For researchers preparing protein samples, Millipore’s PureProteome Protein A and Protein G Magnetic Beads are designed to ensure reproducible isolation of proteins by allowing for total removal of buffers for complete recovery of beads with no sample dilution,” says Wasienko.
Bead-based profiling assays, especially when multiplexed, also allow researchers to miniaturize their experiments. “For academics, many of whom work with limited amounts of reagents to begin with, and who generally perform experiments with small sample volume, the ability to miniaturize easily and effectively is a major benefit,” says Sara Howland, product portfolio leader for drug discovery reagents at PerkinElmer. “This is particularly true when research is being performed with expensive or otherwise limited-availability antibodies – decreasing costs by reducing sample volumes necessary goes a long way to enabling research efficiently.”
Profiling proteins
Profiling assays for proteins is the focus of PerkinElmer’s AlphaScreen and new AlphaLISA, which feature the advantage of no wash steps. Obviously, this simple characteristic saves time and money; but it also reduces assay variability, according to Sara Howland, product portfolio leader for drug discovery reagents at PerkinElmer, as well as yielding high sensitivity and the opportunity to use lower-affinity antibodies.
AlphaScreen is already used for applications such as kinase screening, protein-protein interactions, and GPCR signaling. “AlphaLISA is a new technology, based on AlphaScreen, which is much more resistant to interference from compounds like hemoglobin. This allows PerkinElmer to develop no-wash assays in sample matrices like serum and plasma,” Howland says. “We are also seeing an increasing need for enabling technologies to study protein-protein interactions, which is a huge challenge for drug discovery and academic researchers, as there are few technologies that perform this capability reasonably well, if at all.” PerkinElmer hopes that AlphaLISA and AlphaScreen will facilitate the quantification of protein-protein interactions for more researchers.
Assay Designs is aiming to make multiplexing more accessible in their profiling assays. “Our new MultiBead™ multiplex assay is our vehicle for integrating multiplex capabilities with Assay Designs' 16 years of experience in creating high-quality ELISAs,” says Vespremi. “Our proprietary conjugation chemistry allows us to perform both a sandwich and a competitive immunoassay within the same matrix to quantify large and small molecules at the same time, something which is unique to the MultiBead™ platform. Furthermore, data analysis is performed using our free MultiBead™ Analysis software, so we're helping make multiplex protein analysis accessible and affordable for many more researchers.”
Profiling nucleic acids
A promising new area of bead-based profiling assays is its application to microRNAs, short nucleic acids that are vital for many regulatory functions. “MicroRNA profiling is becoming extraordinarily important for accurately diagnosing a wide range of cancers, and appears to be useful for treating some cancers,” says David Smith, associate director of scientific awareness at Luminex. “Each type of tissue, and each type of tumor, has a unique microRNA profile, making these profiles more accurate and valuable than DNA or proteins.” He cites an example from the lab of Richard Neubig at the University of Michigan, which has developed a new assay that quantifies kinase activity. “Kinase activity is normally tightly regulated, and deregulated kinase activity causes a number of diseases, among them several cancers,” says Smith. “Disease management will become more effective because this multiplexing assay will allow us to determine whether a specific patient is responding to the [therapeutic] drug.”
Luminex’s xMAP® platform, a basis for other vendors’ assays as well, provides the opportunity to measure 100 species in one well of a 96-well plate. Their technology involved the use of 100 sets of tiny color-coded beads whose surface chemistry allows the unique attachment of an antibody, receptor, oligonucleotide, or peptide (100 of them). Each tag is then associated with a set of uniquely colored beads. The beads are passed single file through a space where, individually, each is excited by two lasers that determine precisely what color the bead is (via the bead’s internal dye), and the extent of binding of its attached tags to the species of interest (via reporter dyes). “In the near future,” says Smith, “Luminex will be launching a next-generation instrument that will increase our multiplexing capacity from 100-plex to 500-plex.”
Exiqon has applied their own locked nucleic acid (LNA) technology to the xMAP® platform in their FlexmiR™ microRNA line, a bead-based profiling system that can accomplish microRNA expression profiling in 4 hours, according to Exiqon. The assay is faster and more convenient because it uses the total RNA sample, eliminating enrichment protocols. Furthermore, Exiqon’s LNA capture probes allow greater specificity when differentiating between small nucleic acids that may differ by only one or a few nucleotides.
Marligen Biosciences has also taken advantage of the xMAP® platform for their nucleic acid profiling assays. “We have assays that detect microRNA expression levels, DNA binding of activated transcription factors, and assays that detect genetic variants including SNPs, and gene insertions and deletions,” says Fiona Coats, their VP of business development. “We now offer over 170 individual assays, which can be bought as pre-mixed disease-specific panels or can be custom-mixed to create a multiplex that meets the customer’s specific research needs.” Coats says that historically, companies that develop bead-based profiling assays have been more active in protein profiling, but this may change. “There is a wealth of molecular applications that can be formatted as bead-based assays,” she says. “I believe this will be the new wave for these assays, especially in clinical research and molecular diagnostics, where smaller discrete panels of biomarkers for specific diseases can be profiled on large numbers of patient samples in a fast, efficient way using bead-based technologies.” With future technologies on the horizon, bead-based profiling assays are already proving their worth.