by Alan Dove, PhD
| Figure 1. The Verigene platform can detect single nucleotide polymorphisms associated with inherited diseases. (Image courtesy Nanosphere.) |
Call it polymerase envy — that nagging insecurity protein chemists have had for the past 15 years or so — as they watched their gene jock colleagues apply the astonishingly versatile technique of PCR to virtually every experimental problem. Besides its now-considered-mundane capabilities, such as detecting specific gene sequences at single-molecule sensitivity, PCR underlies everything from diagnostic and forensic tests to the latest genomic research.
Watching this technical revolution from the sidelines, protein chemists are mostly stuck with tools that their forefathers would have recognized. The enormous advances in mass spectrometry have certainly made some analyses easier, but many protein chemistry experiments, and nearly all protein diagnostic tests, still employ much more primitive implements: ELISA and her children, immunohistochemistry, and slightly modernized versions of classical chromatography techniques.
It wasn’t supposed to be like this. As early as 1992, Charles Cantor and his colleagues invited protein chemists to join the newly hatched PCR movement, using a technique called immuno-PCR.(1) In the initial design, immuno-PCR essentially replaced the enzyme in an ELISA with a DNA oligonucleotide. The DNA-linked antibody could be used in the same way as a conventional antibody, but once it bound its antigen, the researchers could detect it using PCR. The thermal cycler’s legendary amplification capability promised to bring single-molecule sensitivity to the protein world.
So why didn’t it? “There are very significant technical difficulties in coupling a piece of DNA to an antibody, it seems, and making it accessible to the PCR reaction, and getting a good signal-to-noise ratio in the test,” explains Christopher Stanley, Ph.D., director of Iseao Technologies in London. Stanley adds that “There are many groups who’ve tried this, and we’ve all had difficulties.”
A new kind of hook-up
| Figure 2. An artist’s rendition of a nanoscale gold bead decorated with DNA “barcodes,” which can tag target proteins during a co-precipitation procedure. (Image courtesy Nanosphere.) |
In the years since immuno-PCR was first described, several companies have tried to develop more practical versions of the technique, but none have succeeded to date. Iseao, a small start-up technology firm, is hoping to have better luck. Instead of pegging its hopes on a purely PCR-based method, the company uses a hybrid approach.
“We decided to go and use the existing immuno-technologies, which are enzymatic labels,” says Stanley. Most experts seem to agree that linking the DNA molecule to the antibody is the most troublesome step in immuno-PCR. Rather than trying to fix that problem, Iseao stepped around it by linking alkaline phosphatase enzymes to the antibodies instead. The enzyme-antibody linkage is ubiquitous in ELISA protocols, and relatively simple.
Instead of triggering a colorimetric assay, though, the enzyme in Iseao’s system removes a phosphate from a specially designed secondary DNA probe, allowing the probe to be amplified by PCR. “So we’re using alkaline phosphatase in its standard form, which is easy technology and well established ... and giving it a DNA substrate and subsequently producing a PCR reaction. It is the most sensitive method I know for measuring the activity of alkaline phosphatase,” says Stanley.
The researchers have applied the same general concept to other types of assays as well. For example, they have designed special DNA substrates for the enzyme ATP ligase. These probes can be amplified by PCR, but only after the ATP-dependent enzyme processes them, so the system is a highly sensitive assay for the presence of ATP. “It’s an alternative to luciferase. There’s a big industry market out there for ATP detection, and what we’ve done is detect ATP using a PCR machine rather than a luminometer,” says Stanley.
Designing a whole new technique just to move an assay from one benchtop machine to another may seem quixotic, but Stanley and his colleagues argue that it will expand the reach of ATP-based assays. PCR has made thermal cyclers almost universally available in laboratories, so porting established assays from more exotic systems into this huge installed base could be quite profitable.
Hybrid enzyme-PCR reactions could also enable entirely new types of assays. With DNA substrates for a different ligase, called NAD ligase, Iseao hopes to provide a definitive system for sensitive detection of bacteria.
The NAD ligase enzyme is evolutionarily conserved throughout eubacteria, but it isn’t found in any other branch of life. If it is present in a sample, it will process the company’s new probe and make it available for PCR amplification, signaling the presence of any species of bacteria. “We’re currently developing a product that detects bacterial contamination in platelets in the blood fraction, which is a really perfect application for us,” says Stanley. In blood, he points out, the specific type of bacterium is often irrelevant: “We’re not interested in which cell it is, we’re interested in showing a level of contamination.”
Size matters
| Figure 3. An illustration of the principles in Iseao’s ligase-mediated ATP amplification assay. |
Despite the ubiquity of thermal cyclers, not everyone sees PCR as a panacea. Indeed, a few companies and researchers are actively promoting alternatives to PCR-based detection, for both proteins and nucleic acids. “[Our technique] is a PCR-less detection system that allows one to detect nucleic acids at relatively high sensitivity, but in a massively multiplex manner,” says Chad Mirkin, Ph.D., director of the International Institute for Nanotechnology at Northwestern University in Evanston, IL.
In place of PCR amplification, Mirkin’s system uses nanoparticles. Users first mix their sample with nanometer-scale beads linked to a probe that binds their target molecule, and then add secondary nanoparticles festooned with DNA “barcodes.” The number of sandwiched nanoparticles that precipitates from the solution is a direct measure of the target’s concentration, and because each analyte is coded with its own DNA sequence, a single assay can measure many targets at once.
Nanosphere, an Evanston-based company Mirkin founded, is now commercializing the technique as part of its Verigene platform. So far, the available Verigene tests focus exclusively on DNA, with products that can detect single nucleotide polymorphisms associated with inherited diseases, and a line of environmental testing systems to detect the DNA of biological weapons. Though detecting proteins with barcodes may be slightly more challenging, Mirkin says it’s definitely on the agenda: “Within the next few years, you will see high-sensitivity protein detection throughout the market, based upon the barcode assay or variants of it.”
For the protein assays, researchers will still have to link antibodies or other ligands to a probe, but promoters of the technique say that the procedure is not as hard as the DNA-antibody linkage that hobbles immuno-PCR. “We found that it’s actually fairly straightforward,” says Bill Cork, chief technology officer at Nanosphere. He concedes that each new protein target requires some tinkering, but doubts that will be a deal breaker. “We were able to make stable probes for a number of different applications ... there’s an effort you need to go through, but it’s not years of effort,” says Cork.
Once the nanoparticle probe is ready, the assay is relatively fast and exquisitely sensitive, often three or more orders of magnitude better than the best ELISA. That could open entirely new avenues of disease monitoring. “The beauty of the barcode assay is the high sensitivity, and the fact that you can track [biomarkers] that there’s no way you can track with ELISA,” says Mirkin.
A rolling circle gathers no users
While highly sensitive protein assays and diagnostic tests certainly sound promising, the path Iseao and Nanosphere are following is not exactly paved with successes. Indeed, immuno-PCR is just one of several clever techniques researchers have developed for this application over the years.
One previous contender, called rolling circle amplification, even had some of the same benefits as Nanosphere’s system. “We were able to detect single molecules in serum, and multiple analytes by using different antibodies labeled with two different oligos ... it worked like gangbusters,” says David Ward, Ph.D., who developed the technique while he was a professor at Yale University in New Haven, CT.
In a rolling circle assay, researchers attach a circular DNA probe to their test antibody, then add oligonucleotides and a polymerase, prompting a continuous replication reaction around the circle. A long, single molecule of DNA then reels out like paper off a roll, repeating the circle’s sequence to amplify the signal. Because the circles and their amplified products stay attached, the technique can even be used for assays that flummox PCR, such as immunohistochemistry, and the continuous replication doesn’t even require a thermal cycler.
Despite all these selling points, Ward, who is now deputy director of the Nevada Cancer Institute in Las Vegas, NV, has watched his rolling circles grind to a halt. “Like immuno-PCR, rolling circle in terms of its utilization in any kind of diagnostic testing modality or any clinical activity, there’s virtually nothing ... it’s sort of in my mind gone down the tubes as a long-forgotten art,” says Ward.
Still, the newer assays, with their motivated corporate backers, seem poised to accomplish what neither classical immuno-PCR nor rolling circles could: bring PCR-like capabilities to the protein world. For protein chemists suffering from polymerase envy, it should be both reassuring and disappointing to know that, as in 1992, the cure is just around the corner. Maybe this time it will make the turn.
References 1. Sano T, Smith CL, Cantor CR, “Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates,” Science, 1992 Oct 2;258(5079):120-2.