Although a standard blood test in clinical laboratories, the enzyme-linked immunoadsorbent assay or ELISA, falls short when it comes to detecting low concentrations of breast cancer, Alzheimer's, and Creutzfeldt-Jakob Disease (CJD) biomarkers. Recently, researchers at the University of Pennsylvania in Philadelphia developed a new enzyme amplification method, known as the Florescent Amplification Catalyzed by T7 polymerase Technique or FACTT, which lowers the limits of detection by 100,000 fold in comparison to ELISAs. This issue of Strategies examines this new technique and what clinical laboratory directors need to know about potential applications.
There are two primary differences between the ELISA and FACTT techniques, according to Mark Greene, MD, PhD, Professor of Medical Science at the University of Pennsylvania (Philadelphia), and lead author of the recently published paper describing the new amplification technique in the online version of Nature Medicine (12 March 2006; doi: 10.1038/nm1378). ELISA, unlike the FACTT technique, uses an enzyme specifically linked to an antibody as a marker for identifying specific proteins. The new immunoassay method incorporates some tools from nucleic acid analyses and some other twists (see figure, below).
“The first difference is that the technique uses a reusable substrate in the form of a double strand of DNA with a promoter and enhancer built into it and is attached to the antibody,” Greene explained. “And we use an enzyme in constant amount in the subsequent dilutions. So the actual configuration of the test is very different.” The second difference involves excess substrate. In ELISA, as the assay is diluted down, the excess substrate can cause substrate inhibition, which doesn't occur in the FACTT method, he explained.
Because of its remarkable detection capability, the FACTT method has created quite a stir, and was even profiled last month on ABC News. ELISA tests can only detect some proteins when they are in high concentration. But many current diagnostic targets only exist in low concentration until a disease is present. One example is proteins associated with the Her2/neu gene, which was initially identified by Greene's laboratory in the early 1980s. While a low-concentration molecule, Her2/neu is overexpressed in more than 30% of primary breast, ovarian, and pancreatic tumors, and higher blood concentrations of the Her2/neu proteins are associated with a lower response rate to chemotherapy, as well as a shorter survival time after relapse, although an important aspect of Her2/neu overexpression is that these patients tend to respond to Herceptin therapy.
In Greene's lab, researchers compared detection of Her2/neu between ELISA and FACTT, using mouse models that were injected with cancer cells that overexpress the Her2/neu protein. While ELISA didn't detect the protein in mouse blood until the animals had developed tumors that had reached an inoperable size, the FACTT technology detected Her2/neu in some mice when the tumors were barely visible and only two days after the cancer cells had been implanted. These results suggest that FACTT can detect signals from cancerous tumors early enough so treatment or prevention efforts can be effective, according to Greene.
FACTT in the Lab
The FACTT technique—as well as the application in measuring circulating Her2neu—is a major advance in ELISA technology and potentially could have a large impact on measurements in the clinical laboratory, said Lawrence Silverman, PhD, Professor of Pathology at the University of Virginia in Charlottesville . “I think that it's a continuation of the concept of increasing the sensitivity of an assay by an amplification technique,” he added.
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Used with the permission of Mark Green, MD, PhD,
University of Pennsylvania (Philadelphia) .
“What they are really taking is a molecular technique in terms of the T7-polymerase tag and then tying this to the sensitivity and specificity of an antibody technique. That melding of technology is exploiting the strengths of both techniques. If you follow immunoassays, and how they've developed and how they went from the radioimmunoassay through immunoassays that were non-radio isotopic, sandwich assays, which is what we have here, it's a logical extension.” Daily clinical applications are probably two to three years down the line, and the Penn group is currently organizing clinical trials to validate FACTT. But aside from clinical applications, Silverman, whose lab studies cystic fibrosis, has talked with his colleagues about other research-oriented applications. One colleague does immunohistochemical staining, and is looking for particularly sensitive assays and detection methods allowing him to see very small concentrations of antigens. “He is looking for more sensitive detection techniques for anything that uses an antibody,” he explained. “So immunohistochemical staining, Western blots, etc.—all these things in the research world are affected by this.”
Moving forward using FACTT, Greene and his colleagues are finishing research on detecting prions, in addition to launching clinical trials for FACTT. The Penn team will develop prototypes for these tests, which the university will then license to manufacturers. “We can detect prions in the blood and we're working on a new test for melanoma and we're just starting ones for lung cancer and colon cancer,” said Greene. “In terms of other clinical applications, we are looking at utilizing this for TNF-alpha detection for autoimmune disease and we believe that FACTT can be used as a readout for both inflammatory and perhaps even some neuropsychiatric problems.”
For more information:
A full copy of Greene's article is available for a fee through the Advance Online Publication section of Nature Medicine 's Web site: www.nature.com/nm/journal/vaop/ncurrent/index.html .
Julie McDowell is the Editor of Strategies. She can be reached by email.