It’s difficult to overstate the power of an antibody in a research laboratory. Antibodies allow researchers to identify and localize proteins of interest, isolate cells specifically expressing those proteins, and to pluck those proteins—as well as any interacting partners—from complex mixtures for deeper biochemical analysis.
With literally hundreds of thousands on the market, antibodies don’t simply make such experiments convenient; they make them possible. With the exception of other classes of binding molecules, such as aptamers, antibodies are the only reagents researchers can use to identify and quantify a given protein in the cellular milieu.
“Antibodies, to me, are the tool of the trade of a biologist as a golf club is to a golfer,” says Robert Balderas, vice president of biological sciences at BD Biosciences (Franklin Lakes, N.J.), which offers a diverse portfolio of antibodies for life science research. “Without them, we cannot do our work.”
A bibliometric analysis published this past February in Nature highlights the influence of research tools such as antibodies in directing research.1 In an essay calling for the development of banks of chemical probes and antibodies for the human proteome, Aled Edwards of the University of Toronto, and colleagues counted publications involving nuclear receptors, a family of small-molecule-binding transcription factors. More than 30 such proteins exist, yet research has focused mostly on just eight—the same eight, in fact, for which chemical probes exist. “In short,” the authors wrote, “where high-quality tools are available (often commercially), there is research activity; where there are no tools, there is none.”
Once the exclusive domain of immunologists, who used them to characterize cells of the hematopoietic lineage, antibodies are now used across the life sciences, including in neuroscience laboratories. Whether studying neural cell differentiation from stem cells, the etiology of neurologic disease, or screening compound libraries for potential therapeutics, neuroscientists rely on antibodies to lend specificity to their science. Antibodies, says Balderas, “are the most exquisite and most valuable resources of biologists to help them make discoveries.”
Take Larry Goldstein, for instance. As director of the University of California, San Diego, Stem Cell Program and a Howard Hughes Medical Institute investigator, Goldstein studies the differentiation of embryonic stem cells into the cells of the nervous system—neurons, astrocytes, glial cells, and so on. His team is well versed in the subtle art of stem cell manipulation, and can coax them down desired pathways.
The problem is that the cell populations that result from such treatment are not homogeneous; instead, they represent a mixture of different neuronal cell types. That complicates the team’s analyses, because they cannot be sure which cell types produce what proteins. “If you have a mixture, it’s hard to know what’s going on in any one cell type,” he explains.
What the team needed was a way to specifically identify and enrich only those cells in the mixture that they wanted to study. Unfortunately, no such molecular signature had been identified. So Goldstein’s team, in collaboration with researchers at BD Biosciences, set out to identify a set of antibodies that could specifically enrich the precursors of neural cell lineages—so-called neural stem cells.
Using fluorescence-activated cell sorting studies of cells taken at various points in the differentiation process and a panel of 190 fluorescently tagged antibodies to different cell surface antigens, the joint BD-UCSD team determined that cells expressing both CD184 and CD4, but neither CD271 nor CD44 could differentiate into any cell in the neural development pathway. They were neural stem cells. The team also identified molecular signatures for neurons and glial cells specifically.
The full BD Biosciences antibody panel, called the BD Lyoplate Human Cell Surface Marker panel, contains 242 antibodies, each of which is independently available for assembly of custom panels.
For Goldstein, these reagents constitute an essential technology. “We couldn’t think of another way to do it,” he says, “So for us, it’s huge.” But it is just a tool, he adds. Now the goal is to put that tool to work, in this case, to pull out specific cell populations for studies into the biochemistry of neurodegenerative diseases.
Neuroscience-specific antibodies are good for more than just cell purification, though. At Bioo Scientific (Austin, Texas), researchers are using antibodies to target specific cell populations for genetic knockdown using what the company calls T3 antibody technology.
T3 technology couples a targeting reagent—an antibody—to a payload, in this case, the regulatory RNAs called short-interfering RNAs and microRNAs. To do that, Bioo Scientific couples commercial antibody preparations to positively charged proteins via a chemical linker. Those positive proteins act as a “sink” for negatively charged short RNAs, says Masoud Toloue, the company’s director of genomic research. By targeting those antibody-RNA complexes via specific neuronal antigens, researchers can target their payloads directly to cells of interest.
“By coupling siRNAs or microRNAs to an antibody, you are able to knock down the gene of interest in the subpopulation of cells you are trying to focus on,” Toloue says.
Bioo Scientific has used T3 preparations in mice to block gene expression in specific cell populations in living tissues. They have not yet targeted neuronal populations in living animals, but they have done so in neural cell cultures, knocking down gene expression in targeted cells by as much as 95%, Toloue says.
Other companies develop antibody-based reagents to support drug discovery efforts. Enzo Life Sciences (Farmingdale, N.Y.), for instance, in collaboration with the Spinal Muscular Atrophy (SMA) Foundation recently launched an ELISA kit for the “survival motor neuron” (SMN) protein, a biomarker implicated in spinal muscular atrophy. “The SMN ELISA kit addresses a critical gap in SMA research and is expected to significantly accelerate SMA therapeutics development,” said Karen Chen, chief scientific officer of the SMA Foundation.
Enzo also offers dyes for visualizing protein aggregation in cells and tissues—a molecular event associated with such neurodegenerative disorders as Alzheimer’s, Huntington’s, and Parkinson’s diseases. Though not antibody-based, the company’s ProteoStat Aggresome Detection Kit and ProteoStat Amyloid Plaque Detection Kit can be used to screen chemical libraries for molecules that can block or reverse aggregation events, says Enzo’s chief science officer Wayne Patton. The dyes can also be combined with fluorescently labeled antibodies in immunocolocalization studies to confirm the nature of the aggregating protein or to identify associated proteins, Patton adds.
In Miami, Vance Lemmon uses antibodies, and a high-throughput microscopy system, to identify genes and compounds that can influence neurite growth.
Unlike most cells in the body, neurons send out long cellular processes from their cell body. In vivo, those processes are called axons and dendrites; in a culture dish, they are termed “neurites.” Neurite growth is critical to recovery from neurologic injury, and Lemmon, the Walter G. Ross Distinguished Chair of Developmental Neuroscience at the University of Miami School of Medicine, is searching for molecules that can influence that process.
Lemmon says he’s been pursuing the problem for years. In 2004, his lab published a paper in which they expressed mutants of one particular protein called L1 in primary neurons. To quantify results in those cells, his team manually traced and measured with software the extent of neurite growth in 2,000 transfected cells in response to different mutants. “It’s really boring to hand trace neurons, really annoying,” Lemmon says.
Fortunately, he doesn’t have to do that anymore. Now, using fluorescently labeled antibodies and a high-content imaging (or screening) system called the Thermo Scientific ArrayScanVTI HCS Reader, he can quantify hundreds of thousands of neurons per day.
The ArrayScan VTI is essentially an automated microscope and analysis platform that can image each well of a microplate and quantify specific morphologic parameters, like total neurite length, or longest neurite length, based on staining. “Really high-content imaging puts fluorescently labeled antibodies to work so we can quantify cellular processes like never before,” says Mark Collins, director of global marketing for Cellomics at Thermo Fisher Scientific (Pittsburgh, Pa.).
To leverage that work, Lemmon’s team runs multiplexed experiments—transfecting cells with a fluorescent marker (to identify transfected cells) and combining that with stains for nuclei and antibodies to different neuronal markers, such as beta-3 tubulin and tau. Such antibodies are widely available commercially from such companies as Santa Cruz Biotechnology and EMD Millipore, and their catalogs can be voluminous. Abcam, for instance, offers nearly 60,000 primary antibodies alone. Epitomics, which concentrates on rabbit monoclonals, boasts 6,800, with another 75 to 90 coming out every month, according to marketing manager Sung Lee. Lemmon prefers homemade antibodies because commercial products would be “prohibitively expensive,”at his team’s throughput level.
In one case, Lemmon’s team used the instrument to screen some 4,000 compounds for those capable of extending neurites in an in vitro model of the glial scarring that occurs after spinal cord injury. In another study, Lemmon and his collaborators used the CellomicsVTI to identify genes that inhibit neurite growth. From a library of 854 different cDNAs, they identified a family of transcription factors called KLFs that, when altered, restored axon regeneration in vivo.
According to Lemmon, in both cases, the combination of specific antibodies and automated imaging were absolutely essential to the project’s success. “There would have been no other way to do this,” he says. “You couldn’t do this one at a time, it would take decades.”
Indeed, for the researchers that rely on them, both within neuroscience and beyond, antibodies have become an enabling technology. “It’s a tool, a way of getting purified cells so we can study them,” says Goldstein. “But it’s a terrific tool.”
About the Author
Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in cell and molecular biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
1. Edwards AM, et al. Too many roads not taken. Nature. 2011;470:163-5.
2. Yuan SH, et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PloS ONE. 2011; 6(3):e17540.